 |
INTRODUCTION |
Insulin-like growth factor-I
(IGF-I)1 is a
growth-promoting polypeptide with diverse cellular functions. IGF-I is
involved in the growth and differentiation of various cell types such
as muscle and adipocytes (1-3). Although liver is the primary source of circulating IGF-I, significant expression of this growth factor is
seen in various tissues including brain, where it is known to exert
autocrine and paracrine functions (4, 5). IGF-I has been shown to
stimulate neurite outgrowth and promote survival of neurons in culture
(4, 5).
IGF-I exerts its cellular effects through its type I IGF receptor which
resembles the insulin receptor in structural as well as functional
aspects (reviewed in Refs. 6 and 7). This heterotetrameric
transmembrane glycoprotein consists of two
- and two
-subunits.
The
-subunit has intrinsic tyrosine kinase activity that is
stimulated when IGF-I binds to the
-subunits. The receptor tyrosine
kinase in turn phosphorylates intracellular substrates such as insulin
receptor substrates 1 and 2 and Shc (7, 8). The tyrosine
phosphorylation sites on these docking proteins recruit Src homology
2-containing proteins such as Grb2, Nck, Crk, SHP2, and the p85 subunit
of PI 3-kinase. From this intermediary complex of signaling proteins,
two significant pathways emerge. One pathway activates extracellular
signal-regulated kinase 1/2 (ERK1/2) through Ras/Raf/MEK, and another
pathway proceeds through PI 3-kinase. IGF-I has been studied
extensively in the PC12 cell line, a model of neuronal tissue. In these
cells, IGF-I promotes growth and proliferation, primarily via
activation of the ERK pathway (9). For the prevention of apoptosis,
IGF-I requires the PI 3-kinase pathway (10).
One of the common nuclear targets of tyrosine kinase signaling cascades
is CREB, the Ca2+/cyclic AMP response element-binding
protein. CREB is a 43-kDa nuclear transcription factor belonging to the
CREB/ATF family (11). Activation of CREB by forskolin, a potent
stimulator of cAMP, stimulates PC12 cell differentiation to a
sympathetic neuron-like phenotype with neurite extension (12, 13). NGF
and IGF-I regulation of CREB is essential for neuronal plasticity, full
axonal development, memory consolidation, and neuroprotection (14-20).
IGF-I is known to regulate a number of CREB response element
(CRE)-containing genes including bcl-2 and c-fos
(21, 22). CREB is constitutively expressed, and it binds to the
specific sequence, 5'-TGACGTCA-3' known as CRE. Phosphorylation on the
serine 133 residue of CREB increases its transcriptional activity. This
phosphorylation does not alter the binding of CREB to CRE, but it
increases its association with adapter proteins such as CREB-binding
protein, leading to the activation of transcriptional machinery. CREB
was initially identified as a substrate for PKA and a mediator of
cAMP-regulated gene expression (23). Later studies showed that CREB can
be phosphorylated and activated by multiple signaling pathways
including ERK, protein kinase C,
calcium/calmodulin-dependent protein kinases, and p38 MAPK
(12, 24-26). Thus, diverse signaling pathways, many of which are
activated by IGF-I, are capable of regulating this transcription
factor, which plays a role in neuronal growth and survival.
Chromogranin A is an acidic glycoprotein present in secretory granules
of the neuroendocrine system (27). The promoter region of this gene has
a conserved CRE site, which is essential for transactivation in PC12
cells (28). Chromogranin A, being a physiologically relevant
CRE-dependent gene, can serve as a read-out for
IGF-I-mediated gene regulation in PC12 cells. We recently demonstrated
an ERK-dependent CREB activation by insulin in Hep-G2 and
3T3-L1 cell lines (29). Since CREB is capable of regulating many
important functions in neuronal tissues, we investigated whether CREB
was important for IGF-I-mediated gene regulation in PC12 cells. The
objectives of this investigation were to (a) examine whether
IGF-I stimulates the phosphorylation and transcriptional activation of
the nuclear transcription factor CREB in PC12 cells, (b)
gain insight into the mechanism by which IGF-I-mediated signal transduction pathways lead to the activation of CREB, and
(c) assess the impact of IGF-I on the neuronal specific
CRE-dependent gene chromogranin A.
We demonstrate that IGF-I increases CREB serine 133 phosphorylation and
transcriptional activation of CREB reporter systems and the neuronal
specific gene chromogranin A through at least three pathways: PI
3-kinase, MEK/ERK, and p38 MAPK. The novel finding of this study is the
contribution of the p38 MAPK-mediated signaling pathway to the
regulation of CREB-dependent gene expression by IGF-I.
 |
EXPERIMENTAL PROCEDURES |
Materials--
PD98059, wortmannin, and rapamycin were purchased
from Biomol (Plymouth Meeting, PA). SB203580 and SB202190 were obtained from Calbiochem. Cell culture media and supplies were from Life Technologies, Inc. and Gemini Bio Products, Inc. (Calabasa, CA). The
plasmid for the expression of the chimeric protein (Gal4-CREB) consisting of the DNA binding domain of Gal4 and the transactivation domain of CREB and the expression vector for Gal4-CREB protein with
serine to alanine substitution at position 133 were a generous gift
from Dr. William J. Roesler (University of Saskatchewan, Saskatoon,
Canada). An expression vector for the luciferase reporter gene driven
by the enhancerless thymidine kinase (TK) promoter linked to four
copies of Gal4 regulatory sequence (pGal4-TK-Luc) was
provided by Dr. James Hoeffler (Invitrogen, San Diego, CA). Three
constructs of mouse chromogranin A promoter linked to luciferase in the
promoterless luciferase reporter vector pXP1 were provided by Dr.
Daniel O'Connor (San Diego, CA). The full-length promoter pXP1133
contained 1133 bp in the 5'-flanking region. The CRE-containing truncated promoter, which maintains the minimal neuroendocrine specificity, is in pXP77; the CRE-mutated version of pXP77 is pXPM41.
Constitutively active and dominant negative Ras and Raf-1 were obtained
from Arthur Gutierrez-Hartmann (University of Colorado Health Sciences
Center, Denver, CO) and Ulf Rapp (Strathlenkunde, Germany). For the PI
3-kinase, SR
-wild type p85, and SR
-
p85 were provided by Dr.
Masato Kasuga (Kobe, Japan). The constitutively active form of MAPK
kinase 6 was obtained from Joel Raingeaud (Institut Curie, Orsay,
France), and the pcDNA3-p38
was provided by Jiahuai Han (San
Diego, CA). The luciferase assay kit was purchased from Analytical
Luminescence Laboratory (San Diego, CA). Antibodies specific for CREB,
P-CREB (Ser133), and phospho-ATF-2 and the ATF-2 fusion
protein were obtained from New England Biolabs (Beverly, MA). Antibody
to p38 MAPK (C-20) was obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA), and the dual phosphorylation site-specific antibody
to p38 MAPK was a gift from Dr. Eric Schaefer (Promega). Plasmids for transfection experiments were purified using Qiagen's (Valencia, CA)
Maxi kit. All other fine chemicals were purchased from Sigma.
Cell Culture--
Rat pheochromocytoma (PC12) cells (provided by
Dr. Gary Johnson (Denver, CO) and Drs. Derek LeRoith and Marcelina
Parrizas (NIDDK, National Institutes of Health, Bethesda, MD) were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum, 5% heat-inactivated horse serum, 100 µg/ml
streptomycin, and 100 microunits/ml penicillin at 37 °C. The cells
were cultured in 60-mm dishes for immunoblotting experiments and in
6 × 35-mm wells for transfection studies. Medium was changed
every second day. Confluent cell cultures were split 1:4 and used for
the experiments 4 days later. The cells were fasted for 5 h by
maintaining them in the medium containing 0.1% fetal bovine serum and
0.05% heat-inactivated horse serum before treatment with growth
factors and other agents in the experiments for measuring CREB
phosphorylation. The stock solutions of pharmacological inhibitors such
as PD98059, wortmannin, and SB203580 were prepared in Me2SO
at a concentration of 1000-fold, so that when they were added to the
culture medium, the concentration of Me2SO was below
0.1%.
Immunoblotting--
Immunoblotting for CREB, phospho-CREB, p38
MAPK, and dual phospho-p38 MAPK were carried out as described
previously (29, 30). PC12 cells cultured in 60-mm dishes were fasted
for 5 h before each experiment. After preincubation with
inhibitors for 30 min and incubation with growth factors for
appropriate duration, the cells were washed twice with ice-cold PBS,
and total cell lysates were prepared by scraping the cells with 200 µl of 1× Laemmli sample buffer containing 100 mM
dithiothreitol. The proteins were resolved on 12% SDS-polyacrylamide
gels and transferred to polyvinylidene difluoride membranes. The blots
were blocked with TBST (20 mM Tris-HCl, pH 7.9, 8.5% NaCl,
and 0.1% Tween 20) containing 5% nonfat dry milk (blotting grade) at
room temperature for 1 h. The blots were then treated with the
primary antibody for P-CREB in TBST containing 5% bovine serum albumin
at 4 °C overnight. After three washes with blocking buffer, the
blots were incubated with anti-rabbit IgG conjugated to alkaline
phosphatase for 1 h at room temperature. This was followed by
three washes with blocking buffer, two washes with 10 mM
Tris-HCl (pH 9.5), 10 mM NaCl, 1 mM
MgCl2, and a 5-min incubation with diluted CDP-Star reagent
(New England Biolabs, Beverly, MA) and then exposed to x-ray film. The
membranes were then stripped in the buffer containing 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM
-mercaptoethanol and reprobed with antibody for CREB by a similar
procedure. The intensity of the bands was quantitated by scanning. The
extent of CREB phosphorylation was measured by calculating the ratio of
P-CREB and CREB bands.
For phospho-p38 and p38, the blots were incubated with the primary
antibody against dually phosphorylated p38 (Promega anti-active p38;
140 ng/ml) in TBST for 1 h at room temperature. After three washes
with TBST, the blots were incubated with anti-rabbit IgG conjugated to
horseradish peroxidase for 1 h at room temperature. This was
followed by three washes in TBST and incubation with diluted ECL
chemiluminescent reagent for 1 min. The membranes were then stripped
and reprobed with antibody against p38 (C-20, Santa Cruz Biotechnology;
50 ng/ml).
p38 MAPK Assay--
The cells were treated with IGF-I and
inhibitor as described in the figure legends. After washing the cells
with PBS, 200 µl of ice-cold cell lysis buffer (20 mM
Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM
-glycerophosphate, 1 mM
sodium orthovanadate, 10 µg/ml leupeptin, 500 nM okadaic
acid, and 1 mM phenylmethylsulfonyl fluoride) was added.
The cells were scraped, lysed by sonication, and centrifuged for 20 min
to collect the supernatant. The lysate (300 µg) was mixed with 4 µl
of p38 MAPK antibody overnight at 4 °C. Protein A-Sepharose (20 µl) was added and gently rocked for 3 h at 4 °C. After
centrifugation, the pellet was washed twice with cell lysis buffer and
twice with kinase assay buffer (25 mM Tris (pH 7.5), 5 mM
-glycerophosphate, 2 mM dithiothreitol,
0.1 mM sodium orthovanadate, 10 mM
MgCl2). The pellet was suspended in 30 µl of kinase
buffer with 200 µM ATP and 2 µg of ATF-2 fusion protein
and incubated for 30 min at 30 °C. The reaction was terminated by
the addition of 10 µl of 4× Laemmli sample buffer. These samples
were electrophoresed and immunoblotted with antibody to phospho-ATF-2.
The intensities of the bands were measured by scanning.
Isolation of Total RNA and Northern Blot Analysis--
PC12
cells (90% confluence) were cultured in fasting medium in the absence
and presence of 100 ng/ml IGF-I for 24 h. Total RNA was isolated
from these cells using the Qiagen RNeasy kit. RNA samples were
fractionated on denaturing 1.2% agarose-formaldehyde gels and
transferred to Hybond N+ membrane. The 1.6-kilobase pair XhoI/EcoRI insert of rat chromogranin A cDNA
probe was labeled with thermostable alkaline phosphatase using the
AlkPhos-Direct kit from Amersham Pharmacia Biotech. Hybridization,
washing, and detection by CDP-Star were performed according to
manufacturer's protocol. The blots were stripped and reprobed with
labeled
-actin by a similar protocol. The expression of chromogranin
A was normalized to
-actin expression.
Transfection Procedure--
The PC12 cells were cultured to
60-80% confluence for transfection experiments in 6 × 35-mm
plates. For each well, 2 µg of plasmids and 20 µg of LipofectAMINE
reagents (Life Technologies, Inc.) were used as per the manufacturer's
instructions. The plasmid containing the
-galactosidase gene driven
by the SV40 promoter was included to normalize the
transfection efficiency. DNA and the LipofectAMINE reagent were diluted
separately in 100 µl of serum-free medium without antibiotics, mixed
together, and incubated at room temperature for 30 min. The culture
plates were washed with PBS and 800 µl of serum, and antibiotic-free
medium was added. The 200 µl of the plasmid LipofectAMINE mixture was
then added to each well, and the plates were incubated at 37 °C for
4 h. Then 1.0 ml of high serum medium (20% fetal bovine serum and
10% heat-inactivated horse serum) was added and incubated for
approximately 40 h before induction with growth factors for
luciferase. After 4 h of induction, the cells were washed in PBS
and lysed with 100 µl of reporter lysis buffer. In the case of
chromogranin A promoter constructs, the induction was 24 h after
transfection for a period of 30 h. The cells were lysed by
freezing and thawing, and lysate was centrifuged at 14,000 rpm for 30 min. The supernatant was used for the assay of luciferase and
-galactosidase. Luciferase assays were carried out using the
enhanced luciferase assay kit (Analytical Luminescence Laboratory, San
Diego, CA) on a Monolight 2010 luminometer. The
-galactosidase
assay was performed according to the method of Wadzinski et
al. (31).
Statistical analysis was carried out by Student's t test.
 |
RESULTS |
Dose- and Time-dependent Phosphorylation of CREB at
Ser133 by IGF-I--
The nuclear transcription factor CREB
was phosphorylated in a time-dependent manner in response
to IGF-I. There was a 2.3-fold (p < 0.001) increase in
CREB phosphorylation at serine 133 (Fig. 1A) at 10 min when PC12 cells
were stimulated with IGF-I (100 ng/ml). The phosphorylation returned to
near basal level by 120 min. The protein level of CREB did not change
during this 2-h period in the presence of IGF-I. The time course of
CREB phosphorylation mediated by IGF-I was comparable with that of
insulin in 3T3-L1 fibroblasts as reported earlier (29). The
dose-response curve shows a significant increase in CREB
phosphorylation at 10 ng/ml (p < 0.05) with
dose-dependent increases at higher concentrations (Fig.
1B). We observed an additional band with the antibody
specific for the phosphorylation sequence around serine 133 of CREB.
Serine 63 phosphorylation of ATF-1 is known to be detected by the same antibody as serine 133-phosphorylated CREB, since they are 100% homologous for this consensus phosphorylation sequence (32). The
phosphorylation pattern of ATF-1 was parallel to that of CREB in terms
of both intensity and time course.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 1.
Dose- and time-dependent CREB
phosphorylation mediated by IGF-I. PC12 cells were cultured in
60-mm dishes to near confluence and then maintained in serum-free
medium for 5 h. They were treated with 100 ng/ml IGF-I for varying
periods of time, from 10 to 120 min (A). In another set of
experiments, the fasted cells were exposed to increasing concentrations
of IGF-I for 10 min (B). The cells in both experiments were
washed in ice-cold PBS at the end of incubation period, and the cell
lysates were prepared by the addition of 200 µl of warm 1× Laemmli
sample buffer followed by sonication. The samples containing equal
amounts of proteins were electrophoresed and immunoblotted with the
antibody specific for CREB phosphorylated at Ser133. The
membranes were stripped and reprobed with CREB antibody. Quantitation
of specific bands was done by scanning densitometry. The ratio of
phosphorylated CREB over the nonphosphorylated form was calculated, and
this value for the untreated cells was taken as 1. The results are the
mean ± S.E. of four independent experiments.
|
|
Multiple Signaling Pathways Are Involved in IGF-I-mediated
Phosphorylation of CREB--
To explore the role of different
signaling pathways in CREB phosphorylation, we examined the effect of
PD98059 (an inhibitor of MEK) and wortmannin (an inhibitor of PI
3-kinase) on IGF-I-induced CREB phosphorylation. In addition, we
examined the effects of SB203580 and SB202190, two specific inhibitors
of p38 MAPK (33, 34). This protein kinase has been shown to mediate the
effects of fibroblast growth factor and NGF on CREB phosphorylation. As shown in Fig. 2A,
preincubation of PC12 cells with PD98059 (30 µM) or
wortmannin (100 nM) resulted in a 25-30% decrease in
IGF-I-mediated CREB phosphorylation at serine 133. The addition of 30 µM PD98059 decreased the induction of ERK1/2 MAPK
activity as detected by dual phospho-ERK antibody (Promega, Madison,
WI) (data not shown). The findings with wortmannin clearly indicate
that the PI 3-kinase mediates IGF-I-induced nuclear signaling in
addition to the regulation of cytosolic proteins such as glycogen
synthase. The inhibitors of p38 MAPK, SB203580 (10 µM),
and SB202190 (10 µM) were able to decrease the CREB
phosphorylation stimulated by IGF-I significantly (p < 0.001), suggesting a novel pathway for nuclear signaling of this growth
factor. Rapamycin, an inhibitor of p70 S6 kinase, which is one of the
downstream components of the PI 3-kinase signaling system, had no
significant impact on IGF-I-induced CREB phosphorylation.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of pharmacological inhibitors on
growth factor-mediated CREB phosphorylation. PC12 cells (90%
confluent) were fasted for 5 h and preincubated with 30 µM PD98059 (PD), 100 nM wortmannin
(W), 10 µM SB203580 (SB1), 10 µM SB202190 (SB2), and 10 ng/ml rapamycin (R)
for 30 min followed by incubation with 100 ng/ml IGF-I (A).
In some experiments, the cells were preincubated with combinations of
inhibitors before treating them with 100 ng/ml IGF-I or 50 ng/ml NGF
(B). The cells were washed with ice-cold PBS, solubilized,
and immunoblotted for P-CREB. The membranes were stripped and reprobed
with the antibody to CREB. The inhibitors did not have any significant
effect on CREB phosphorylation and its protein level in control cells
(results not shown). The results are the mean ± S.E. of three
independent experiments.
|
|
The neurotrophic actions of IGF-I are similar to those of NGF in PC12
cells. Therefore, we compared the effects of IGF-I and NGF on CREB
phosphorylation. The partial reduction in NGF-mediated CREB
phosphorylation in the presence of individual inhibitors was similar to
that of IGF-I (data not shown) with the minor difference that PD98059
was more effective than wortmannin in decreasing NGF-induced CREB
phosphorylation. In some experiments, combinations of inhibitors were
shown to block the effects of growth factors completely (Fig.
2B). For example, in the presence of wortmannin (100 nM) and SB203580 (10 µM), IGF-I did not
increase CREB phosphorylation above basal level. The inhibitors PD98059
(30 µM) and SB203580 (10 µM) used together
blocked NGF action. For the remainder of the studies, parallel
experiments were conducted with NGF as a control. The NGF data will
only be presented for selected experiments.
IGF-I Activates p38 MAPK in PC12 Cells--
The experiments with
pharmacological inhibitors demonstrated that multiple signaling
pathways mediate IGF-I-induced phosphorylation of CREB, including ERK,
PI 3-kinase, and p38 MAPK. Growth factors have been shown to activate
p38 in neuronal cell lines (26, 35, 36). Because the pharmacological
inhibition of p38 with the SB compounds suggested a role for p38, we
examined p38 activity in response to IGF-I in these cells. To examine
whether IGF-I activates p38 MAPK in PC12 cells, experiments were
carried out to measure the formation of phospho-p38 MAPK, the active
form of this enzyme. As shown in Fig.
3A, IGF-I increased the
phosphorylation of p38 significantly (p < 0.001) over
the untreated cells in 5 min and slowly decreased over the remaining
2-h incubation period. Sodium arsenite is a known stimulator of p38
MAPK activity, and it serves as a control for the immunoprecipitation
kinase assay. When p38 MAPK was assayed in PC12 cells treated with
IGF-I (100 ng/ml) and sodium arsenite (300 µM) by
immunoprecipitation followed by phosphorylation of ATF-2
phosphoprotein, 78 and 145% increases in the enzyme activity were
observed, respectively. Pretreatment of cells with the inhibitor
SB203580 (10 µM) decreased the stimulated enzyme activity
significantly (p < 0.01).

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Activation of p38 MAPK by IGF-I. PC12
cells (90% confluent) were fasted for 5 h and exposed to IGF (100 ng/ml) for varying time periods (A). The cells were washed
and harvested for immunoblotting with the antibody to phospho-p38 MAPK.
The membranes were then stripped and reprobed with the antibody to p38
MAPK. In some experiments, the fasted cells were preincubated in the
absence ( ) and presence ( ) of 10 µM SB203580 for 30 min followed by incubation with IGF (100 ng/ml) for 10 min or sodium
arsenite for 300 µM for 1 h (B). The
activity of p38 MAPK was measured by immunoprecipitating the cell
lysates with p38 MAPK antibody and phosphorylating ATF-2 fusion protein
followed by immunoblotting phospho-ATF-2. Quantitation of the bands was
done by scanning densitometry. The values are the means of three
observations.
|
|
IGF-I-mediated CREB Phosphorylation Does Not Involve
cAMP-dependent Protein Kinase (Protein Kinase A)--
CREB was
initially described as a substrate for protein kinase A (23).
Therefore, we assessed the role of protein kinase A in IGF-I induced
CREB phosphorylation using H89, a pharmacologic inhibitor that
specifically inhibits this kinase (data not shown). This inhibitor
decreased the formation of P-CREB mediated by dibutyryl cAMP (500 µM) and forskolin (10 µM) significantly
(p < 0.001). H89 did not block IGF-I and NGF-mediated
increases in P-CREB formation.
IGF-I Mediated CREB Phosphorylation Leads to Its Transcriptional
Activation--
CREB phosphorylation at serine 133 is essential for
transcriptional activation, but under some conditions this
phosphorylation is inadequate to drive transcription (37). Thus, it was
essential to determine whether IGF-I-mediated CREB phosphorylation
enhanced its transcriptional activation. For initial experiments, we
used a Gal4-TK-Luc reporter system specific for the transactivation of
CREB. Since endogenous transcription factors do not bind to the
promoter pGal4-TK-Luc, the increase in luciferase activity in the
presence of IGF-I is a measure of the stimulation of the transactivational potency of the Gal-CREB chimeric protein through phosphorylation. PC12 cells were transiently transfected with an
expression vector for a chimeric protein consisting of the Gal4 DNA
binding domain linked to the transactivation domain of CREB and a
plasmid containing the luciferase reporter gene linked to an
enhancerless thymidine kinase promoter and four copies of Gal4-responsive sequences. By this approach, one could eliminate other
CRE-binding endogenous transcription factors binding to the reporter
gene. This permits evaluation of the pathways leading specifically to
CREB activation. IGF-I increased transcription in a
dose-dependent manner to a maximum of 3-fold in this system (Fig. 4A). No transcriptional
activation was noted in the control experiments without the Gal4-CREB
chimeric protein (results not shown). To optimize cell viability after
transient transfection, the cells were maintained in serum during the
induction with growth factors, because PC12 cells undergo programmed
cell death with serum withdrawal. This contributes to the high basal
CREB transcriptional activity and also represents the normal
physiological context. Consistent increases over the physiological
background were noted in response to IGF-I.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Transcriptional activation of CREB by IGF-I
in PC12 cells. PC12 cells were cultured in 6 × 35-mm wells
to around 70% confluence. The cells were cotransfected with
pGal4-TK-Luc, pRSV-Gal4-CREB-341, and pRSV -galactosidase in the
medium containing no serum and antibiotics by the LipofectAMINE
transfection method for 4 h. For each well, 2 µg of plasmids and
20 µg of LipofectAMINE reagent were used. Induction for luciferase
with different agents for 4 h was carried out 48 h after the
initiation of transfection. A, the transfected cells were
incubated with varying doses of IGF-I as indicated. B, in
the transfection protocol for this experiment either pRSV-Gal4-CREB or
pRSV-Gal4-CREB S133A was used, and later the transfected cells were
induced with 100 ng/ml IGF or 50 ng/ml NGF. Cell lysates were prepared,
and the transcription was measured by assaying the luciferase activity
by the procedure described under "Experimental Procedures." In
these lysates, -galactosidase activity was also assayed to correct
for the efficiency of transfection. The transcription mediated by IGF-I
and other agents was expressed as -fold induction over the basal
transcription in transfected but untreated cells. Results are
means ± S.E. of three independent experiments.
|
|
To determine the functional significance of phosphorylation of serine
133 by IGF-I for activation of this transcriptional reporter,
cotransfection experiments were carried out using the expression vector
for Gal4-CREB in which serine was replaced with alanine at position
133. When cotransfected with pGal4-TK-Luc, this mutated fusion protein
did not induce luciferase expression significantly when compared with
the wild type Gal4-CREB (Fig. 4B). Treatment with growth
factors did not further enhance the luciferase expression.
IGF-I-mediated Transcriptional Activation of CREB Parallels the
Regulation of CREB Phosphorylation--
A parallel set of experiments
to those described for CREB phosphorylation in Fig. 2 was undertaken to
determine the contribution of MEK, p38 MAPK, PI 3-kinase, and p70 S6
kinase in the transcriptional activation of CREB. PC12 cells
transfected with pGal4-TK-Luc and pGal-CREB were preincubated with
PD98059 (30 µM), wortmannin (100 nM),
SB203580 (10 µM), and SB202190 (10 µM).
These additions decreased IGF-I-mediated CREB-TA by 27, 44, 31, and
34%, respectively (Fig. 5A).
Rapamycin did not have any effect on the transcriptional activation by
IGF-I. In the case of NGF, significant decreases (25-35%) in
transcriptional activation were exerted by the inhibitors PD98059 (30 µM), wortmannin (100 nM), SB203580 (10 µM), and SB202190 (10 µM) (data not shown).
As with IGF-I, rapamycin had no effect on NGF action. The inhibition of
transcriptional activation by these inhibitors was partial when used
alone. Parallel to our observation of the impact of combined inhibitors
in Fig. 2, IGF-I-induced luciferase production was decreased to the
basal level when the cells were preincubated with wortmannin and
SB203580 together (Fig. 5B). These findings clearly
demonstrate that IGF-I uses novel signaling pathways to increase the
transcriptional activation of CREB in PC12 cells.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
Growth factor-mediated CREB activation
involves multiple signaling pathways in PC12 cells. PC12 cells
cultured in 6 × 35-mm wells were transfected with pGal4-TK-Luc,
pRSV-Gal4-CREB-341, and pRSV -galactosidase for 4 h by the
LipofectAMINE transfection method using 2 µg of total plasmids and 20 µg of LipofectAMINE reagent. After 48 h, the cells were first
exposed to the 30 µM PD98059 (PD), 100 nM wortmannin (W) 10 µM SB203580
(SB1), 10 µM SB202190 (SB2), and 10 ng/ml rapamycin (R) for 30 min and then incubated with 100 ng/ml IGF-I (A) for 4 h. In some experiments, the cells
were preincubated with combinations of inhibitors before exposure to
100 ng/ml IGF-I or 50 ng/ml NGF (B). The activities of
luciferase and -galactosidase were measured in the cell lysates. The
-fold increases in CREB activation by IGF-I and NGF were calculated
after correcting for transfection efficiency. The values represent
means ± S.E. of three observations.
|
|
IGF-I Activates Transcription of CRE-containing Chromogranin A
Promoter Constructs--
Once the transcriptional activating potential
of IGF-I on CREB had been determined using the Gal4-TK-Luc reporter
system, we did a series of experiments to assess the physiological
relevance of CREB activation by IGF-I. For these experiments, we
employed the neuronal specific CRE-containing chromogranin A promoters. The promoter with 1133 bp of 5'-flanking region was stimulated 3.3-fold
by IGF-I, whereas the truncated promoter with the CRE (pXP77) was
activated 2.5-fold. To determine whether this activity was dependent
upon CREB, we did a series of experiments cotransfecting a dominant
negative CREB, K-CREB, as well as the truncated chromogranin A promoter
with the CRE site mutated (Fig.
6A). The dominant negative K-CREB decreased basal and total IGF-I stimulation compared with controls, as did the CRE mutant. Some increase in activity was seen in
response to IGF-I, indicating both CREB-dependent and CREB-independent regulation of chromogranin A by IGF-I. We also noted a
3.7-fold increase in chromogranin A mRNA when PC12 cells were
exposed to 100 ng/ml of IGF-I for 24 h (Fig. 6B),
demonstrating that the endogenous gene and the reporter constructs
respond similarly.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
IGF-I-mediated induction of the chromogranin
A gene in PC12 cells. A, PC12 cells were cultured in
6 × 35-mm wells to around 70% confluence. The cells were
cotransfected with pxP1133, pxP77, pxPM41, and pxP77 with dominant
negative K-CREB and pRSV -galactosidase for 4 h by the
LipofectAMINE transfection method using 2 µg of total plasmids and 20 µg of LipofectAMINE reagent. After 24 h, the cells were cultured
in the absence ( ) and presence ( ) of 100 ng/ml of IGF for 30 h. The cell lysates were prepared, and luciferase and galactosidase
were assayed. Results are means ± S.E. of three independent
experiments. B, PC12 cells (90% confluent) were cultured in
fasting medium in the absence and presence of 100 ng/ml IGF for 24 h. Total RNA was isolated from these cells using Qiagen's RNeasy kit.
RNA 10-µg samples were resolved on formaldehyde-agarose gels and
transferred to Hybond N+ membranes and probed with the chromogranin A
cDNA probe labeled with alkaline phosphatase and detected with the
CDP-Star system. The blots were then stripped and reprobed with labeled
-actin by a similar procedure. Two representative blots from the set
of five are shown here.
|
|
Impact of Constituitively Active or Dominant Negative Ras and Raf-1
on IGF-I-mediated Stimulation of Chromogranin A--
Pharmacological
inhibition of MEK using PD98059 (Figs. 2 and 5) indicated a role
for the ERK1/2 MAPK cascade in IGF-I regulation of CREB activity. To
confirm these data using another strategy, we cotransfected PC12 cells
with a truncated CREB-responsive chromogranin A reporter construct and
plasmids for constitutively active Ras and Raf-1 (pSVRas and pRSV
BxBraf, respectively) or dominant negative Ras (pZCRN17Ras). Activation
of the Ras
Raf
MEK
ERK pathway using the constitutively
active iosforms of either Ras or Raf-1 (Fig.
7A) led to a significant
increase in basal chromogranin A activity, demonstrating responsiveness
to this pathway. IGF-I treatment gave an additional stimulation of
1.5-fold over the high basal level, suggesting that there are other
pathways in addition to ERK1/2 that contribute to the IGF-I response.
Dominant negative Ras decreased basal activity with a restoration
toward basal upon exposure to IGF-I. Taken together, these data support a role for ERK1/2 activation of chromogranin A by IGF-I. They also
suggest that additional pathways are involved.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Activation of chromogranin A promoter by IGF
through multiple signaling pathways. PC12 cells (70% confluence)
cultured in 6 × 35-mm wells were transfected with pxP77 and pRSV
-galactosidase along with indicated plasmids for 4 h by the
LipofectAMINE transfection method using 2 µg of total plasmids and 20 µg of LipofectAMINE reagent. In these transfection experiments,
plasmids for the modulation of signaling pathways involving Ras
(A), PI 3-kinase (B), and p38 (C)
were also included. After 1 day of transfection, the cells were
cultured in the absence ( ) and presence ( ) of 100 ng/ml IGF-I
(A and B) or as indicated (C) for
30 h. The activities of luciferase and -galactosidase were
measured in the cell lysates. The -fold in creases in CREB activation by IGF-I and NGF were calculated after
correcting for transfection efficiency. The values represent means ± S.E. of three observations.
|
|
Role of PI 3-Kinase in the IGF-I-mediated Activation of
Chromogranin A--
The PI 3-kinase inhibitor data suggested that one
of the additional pathways involved PI 3-kinase. As demonstrated in
Figs. 2A and 5A, wortmannin, an inhibitor of PI
3-kinase, interferes with both CREB phosphorylation and its
transcriptional activation by IGF-I. Hence, we examined the effect of
transient transfection of wild-type and dominant negative p85 subunits
(courtesy of Dr. Masato Kasuga, Kobe, Japan) in PC12 cells. The wild
type p85 subunit exerted a small increase in basal and IGF-I-mediated
transcriptional activation, whereas
p85, the kinase-dead PI 3-kinase
isoform, inhibited IGF-I activation of chromogranin A
(p < 0.001). These results demonstrate a PI
3-kinase-dependent activation of chromogranin A in PC12 cells.
MAPK Kinase 6 and p38
Enhance the Activation of Chromogranin A
Promoter by IGF-I--
Previous studies have indicated that, among p38
MAPK isozymes, the
isoform is involved in the hypertrophic action.
Hence, we cotransfected the PC12 cells with p38
and the
constitutively active form of its upstream kinase, MAPK kinase 6, and
examined the promoter activity of chromogranin A (pXp77). The
stimulation of p38
resulted in the increase of basal and
IGF-I-induced chromogranin A promoter activity by 80-90%. This
increase was significantly (p < 0.001) blocked when
the cells were preincubated with the p38 MAPK inhibitor SB203580 (10 µM). The results of this experiment further support the
role of the p38 MAPK pathway in IGF-I-mediated activation of the
nuclear transcription factor CREB.
 |
DISCUSSION |
In this investigation, we demonstrate that IGF-I stimulates
phosphorylation of the nuclear transcription factor, CREB, the Ca2+/cAMP response element-binding protein, at serine 133 in PC12 cells. This post-translational modification leads to an
increase in CREB's transcriptional activity as demonstrated by the
Gal4-TK-Luc reporter system. IGF-I is also capable of regulating
chromogranin A, a neuroendocrine-specific gene, by a
CREB-dependent mechanism. Using specific inhibitors such as
PD98059, SB203580, or wortmannin or by cotransfecting constitutively
active and dominant negative components of the Ras and PI 3-kinase
pathways, we demonstrate that IGF-I-mediated CREB activation proceeds
through three distinct pathways involving ERK, PI 3-kinase, and p38
MAPK. IGF-I is known to exert its actions on cellular proliferation,
survival, and differentiation through the ERK and PI 3-kinase pathways.
In this study, we show for the first time that some of the
CREB-dependent gene regulatory actions of IGF-I proceed
through the p38 MAPK pathway.
In neuronal cells, the nuclear transcription factor CREB plays a
central role in several critical functions. It is important for protein
synthesis-dependent long term memory formation, since targeted mutation of CREB leads to a decrease in long term memory in
mice (14, 15). Hormonal regulation of dentritic spine formation in
cultured hippocampal neurons requires the phosphorylation of CREB (19).
In PC12 cells, a cell culture model of neurons, the CREB/ATF-1 family
of transcription factors are needed for the neurite outgrowth (38).
Dominant negative ATF-1 blocks cAMP-induced neurite formation by
inhibiting cAMP-mediated CREB activation in these cells (38).
Interference of CREB and other ATF family members with E1A viral
antigen also blocks PC12 cell differentiation (13). Additionally, CREB
is critical for the induction of immediate early gene c-fos
by NGF (12). The promoter regions of several neuronal specific genes
such as chromogranin A (CgA) and vgf contain CREB response
elements. Because of the diverse neuronal responses that require CREB,
it is important to understand the specific mechanisms whereby IGF-I, an
important neurotrophin, regulates CREB dependent transcription.
CREB is regulated by multiple factors in PC12 cells including
forskolin, NGF, epidermal growth factor, and
12-O-tetradecanoylphorbol-13-acetate (12, 28). These diverse
stimuli can result in divergent cell fates including proliferation and
differentiation. With respect to the diversity of factors that can
impact CREB- and CRE-regulated transcription, we present important new
information on CREB regulation by IGF-I in PC12 cells. We present
convincing data that IGF-I treatment at a physiologically relevant
concentration leads to transcriptionally important phosphorylation of
CREB at serine 133. Additionally, CREB activation plays a major role in
the IGF-I-mediated regulation of the neuronal specific chromogranin A
gene. We see an impact of IGF-I on both the chromogranin A promoter and
induction of chromogranin A mRNA. The experiments described define
many parallels between NGF and IGF-I for CREB regulation in this cell line. Both agents employ multiple signaling pathways for CREB regulation. From inhibitor studies, it appears that the dominant pathways for IGF-1 are p38 MAPK and PI 3-kinase, whereas MEK is the
dominant pathway for NGF with a contribution from p38 MAPK. This role
of p38 MAPK activation by growth factors is a new and rapidly evolving
area of research. The current data do not permit a detailed comparison
between the IGF-I and NGF, but the differences noted between the two
neurotrophins could provide insight into their divergent impacts on
cell fate, survival, and proliferation.
IGF-I has been shown to have significant neurotrophic actions such as
survival and regeneration of neurons (4, 5). In diabetes, IGF-I
activity is decreased in neuronal tissues, and this could contribute to
the development of diabetic neuropathy (39). IGF-I is being considered
as a potential therapeutic agent in the treatment of neurodegenerative
diseases (40). For these reasons, it is essential to understand the
mechanism by which this growth factor regulates gene expression in
neuronal cells. Our present findings show that IGF-I mediated the
activation of the nuclear transcription factor CREB through multiple
signaling pathways and that this leads to enhanced expression of a
neuroendocrine-specific gene, CgA. We chose to examine CgA because it
is known to be regulated in a CREB-dependent manner in PC12
cells (27, 28). This tool establishes a neuronal context for the
experiments designed to define the important signaling pathways. CREB
is known to bind the promoters of mouse and human CgA (27, 41). In the
present investigation, we observed an enhanced expression of CgA in
IGF-I-treated PC12 cells, as measured by the Northern blot analysis of
mRNA. Further, IGF-I-mediated activation of CREB through multiple
signaling pathways leads to the stimulation of full-length as well as
the CRE-containing truncated promoter of CgA. Studies with dominant negative K-CREB and the promoter containing mutated CRE clearly demonstrate that CREB plays a significant role in mediating IGF-I action. These findings indicate that IGF-I-stimulated CREB activation is involved in the physiologically relevant gene expression in neuronal cells.
Our observation that IGF-1 stimulates the phosphorylation of nuclear
transcription factor CREB through a p38 MAPK is new, and the
physiological relevance remains to be determined. To briefly review the
current understanding of the p38 MAPK family, they are a family of
serine/threonine kinases, activated by dual phosphorylation on
threonine and tyrosine residues. In mammalian cells, three distinct
MAPKs have been identified: ERK1/2, stress-activated protein
kinase/c-Jun N-terminal kinase, and RK cytokine suppressive anti-inflammatory drug binding protein p38 MAPK. The pathways mediated
by c-Jun N-terminal kinase and p38 MAPK have been shown to play a
significant role in stress-mediated signal transduction. The p38 MAPK
is associated with apoptosis, and it opposes the actions of ERK in PC12
cells (30, 42). However, p38 and ERK MAPKs cooperate in the
transcriptional activation of c-fos in response to UV
irradiation (43). Further, p38 MAPK participates in the protein
phosphorylation cascade resulting from activation of growth
factor/hormone receptors. For example, fibroblast growth factor
activates p38 MAPK in SK-N-MC cells, and insulin stimulates this kinase
in L6 muscle cells (26, 44). NGF has been shown to activate CREB
through the ERK as well as p38 MAPK pathway (35). IGF-I stimulates p38
MAPK activity in SH-SY5Y neurobastoma cells (36). In a recent study,
Scrimgeour et al. (45) used a mutant of the IGF-I receptor
in which tyrosines at positions 1250 and 1251 in the carboxyl-terminal
region had been replaced to demonstrate that some of the actions of
IGF-I could involve a third pathway other than ERK and PI 3-kinase
pathways. We support this possibility by showing in this study that
IGF-I does activate CREB by a third pathway involving p38 MAPK.
IGF-I-mediated phosphorylation and activation of CREB decreased
significantly in the presence of SB203580. This pyridinyl imidazole
derivative has been shown to be specific for p38 MAPK, and it did not
have any inhibitory action toward 12 other protein kinases tested
in vitro (34). Several isoenzymes of p38 MAPK have been
identified that are likely to have differential actions (46, 47). Wang
et al. demonstrated in cardiomyocytes that p38
mediates
hypertrophic response, whereas p38
induces apoptosis (48). In a
recent study, this
isoform has been also shown to provide
protective effect against apoptotic signals (49). We have also observed
in the present study an increase of IGF-I-induced chromogranin A
promoter activity when the PC12 cells were cotransfected with p38
and the constitutively active form of MAPK kinase 6.
IGF-I has been shown to activate the ERK and PI 3-kinase pathways in
several cell types including PC12 cells (1, 6, 50, 51). In the present
study, the MEK inhibitor (PD98059) partially decreases the
phosphorylation and activation of CREB mediated by IGF-I. Figs. 2 and 5
demonstrate only a partial, albeit significant, inhibition of
IGF-I-mediated CREB phosphorylation and activation using PD98059.
Cotransfection experiments with constitutively active Ras and Raf-1
demonstrate a role for this pathway in the activation of CgA. However,
IGF-I was able to drive transcription of CgA even in the face of
constitutively active Ras and Raf-1. These data suggest that the Ras
Raf-1
MEK pathway is important but is not the dominant pathway
for IGF-1-mediated CREB activation. In a recent study, however, we
observed that the ERK pathway plays a major role in the
insulin-mediated CREB activation in HepG2 and 3T3-L1 cell line,
suggesting cell-specific variations in signaling pathways. The PI
3-kinase pathway, which is critical for IGF-I-mediated neuronal
survival under stress conditions, strongly contributes to
IGF-I-mediated CREB activation. The importance of PI 3-kinase for IGF-I
activation of CREB is clearly demonstrated by the wortmannin inhibitor
studies. It is further supported by cotransfection of
p85 PI
3-kinase, the kinase-dead mutant, which ablates IGF-I-mediated
stimulation of CgA. Akt, the downstream component of the PI 3-kinase
pathway, is known to regulate the covalent modification of cytosolic
proteins such as glycogen synthase and the proapoptotic protein BAD in
PC12 cells (51, 52). In the present study, we demonstrate the
involvement of this pathway in the nuclear actions of IGF-I. It has
been previously shown that the expression of Bcl-xL and the
bcl-2 are increased by IGF-I (21, 53). These proteins belong
to the Bcl-2 family, which is known to protect the cells from
programmed cell death (21). It is possible that IGF-I-mediated CREB
activation is involved in the regulation of the expression of
bcl-2, since it is a CREB-dependent gene
(54).
To summarize, the data presented in this paper demonstrate IGF-1
activation of CREB, an important transcription factor for neurotrophin
activity. This activation employs signaling pathways mediated by PI
3-kinase and p38 MAPK. Future studies will explore the implications of
each of these signaling pathways for CREB-responsive genes important
for cell cycle regulation, survival, and differentiation.