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INTRODUCTION |
Cytokines play an essential role in the regulation of multiple
cellular functions including cell proliferation, differentiation, and
survival. Most cytokines activate Jak/signal transducer and activator
of transcription (STAT)1
signaling pathways, which are indispensable in the regulation of
various cellular programs (1-4). Jaks are cytoplasmic tyrosine kinase
effectors that are activated by ligand-receptor interactions, leading
to the tyrosine phosphorylation and activation of the cytoplasmic
transcription factors, STATs, which then translocate to the nucleus and
act on target gene transcription. To date, genes for four kinases of
the Jak family and seven mammalian STATs have been identified, which
may be activated individually or in combination (5). However, STAT
pathways are not found exclusively in cytokine signal transduction and
thus are also important for several growth factor-activated tyrosine
kinase receptor-mediated mechanisms.
Recent studies using Jak- or STAT-deficient mice have assigned specific
cellular functions to most of these signaling components (6). For
example, Jak1 has been shown to be essential for signaling by
gp130-containing cytokine receptors (7), both Jak1 and Jak3 are
essential in lymphocyte development, and Jak2 deficiency is lethal due
to defective erythropoiesis (7-10). Similarly, the STATs also have
specific roles in cell regulation. Stat1 is essential for interferon
signaling and innate response to viral and bacterial infection (11),
while Stat5a is associated with the mammary gland development (6). In
contrast, Stat3 is critical for embryonic development; Stat3-deficient
mice die before gastrulation (12).
Interleukin-6 (IL-6) belongs to a cytokine subfamily, whose members
include ciliary neurotrophic factor, leukemia inhibitory factor,
interleukin-11, oncostatin M, and cardiotrophin-1 and which share a
common signal transducing molecule, gp130, in their respective receptor
complexes (13). Unlike tyrosine kinase receptors, IL-6 receptor 
subunit (IL-6R) does not possess intrinsic tyrosine kinase
activities. Rather, binding of IL-6 to IL-6R triggers the
association and dimerization of the signal-transducing component gp130.
As with most cytokines capable of inducing multiple cellular functions,
accumulating evidence suggests that this subfamily of cytokines
signaling through gp130 is involved in neuronal regulation in addition
to its well established functions in hematopoiesis, immune, and
inflammatory responses. Ciliary neurotrophic factor and leukemia
inhibitory factor are involved in the neuronal differentiation and
regeneration as well as cell fate determination (14-17). Although not
as well established, IL-6 has also been implicated as a potential regulatory agent in the nervous system (18, 19). Both IL-6 and its
ligand binding subunit IL-6R are expressed and localized in discrete
areas of the central nervous system (20). In addition, they are
synthesized and secreted in both central and peripheral nervous tissues
in response to inflammatory stimuli or other environmental insults
(21-26). Moreover, it has been shown that IL-6 prevents cell death and
promotes survival in some cerebral neurons as well as cultured
sympathetic neurons (21, 27-31).
IL-6 may also play a potential role in neuronal cell differentiation or
regeneration (18, 19, 23, 32). We have reported that it is capable of
inducing morphological differentiation in PC12 variant E2 cells or in
PC12 cells previously exposed to nerve growth factor (NGF) or basic
fibroblast growth factor (bFGF) (primed) but not native PC12 cells.
Furthermore, a synergistic induction of neurite proliferation and
neuronal specific genes was observed by treatment of either wild type
or variant cells with combinations of subthreshold concentrations of
IL-6 and NGF or epidermal growth factor (EGF) (33). Recent reports by
others also suggest an involvement of IL-6 in morphological
differentiation by different PC12 variants under various treatment
conditions (34-38).
In previous studies, the differentiative response of PC12-E2 or primed
PC12 cells to IL-6 was shown to be largely independent of the
activation of the RAS/ERK pathway (32), which is required for the same
response to NGF and bFGF (39, 40). Instead, it was observed that IL-6
predominantly activates Stat3 and to a lesser extent the Stat1
signaling pathway. IL-6 did induce the tyrosine phosphorylation of ERK,
but the activation was found to be transient and very weak compared
with that produced by NGF, bFGF, or even EGF. In contrast, Ihara
et al. (35) have reported that the Stat3 stimulated by IL-6
in PC12 cells pretreated with NGF (1-2 h) is actually inhibitory of
differentiation and that the positive responses observed are via
gp130-mediated activation of ERKs.
To resolve these conflicting mechanisms and to further evaluate the
contributions of these intracellular signaling pathways to IL-6-induced
morphological differentiation of PC12-E2 cells, PC12-E2 cells
overexpressing dominant negative variants of Stat1, Stat3, and
p21ras proteins were examined. The results clearly demonstrate
that activation of the Stat3, and not the RAS/ERK, pathway is involved in the neurite proliferative response to IL-6. Thus, these signaling pathways act in a substantially similar way and are positive
stimulators of PC12 cell differentiation.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Stable PC12 variant E2 cells characterized by
more rapid responses to NGF and bFGF and markedly more robust responses
to IL-6 were isolated from the parental PC12 line as described
previously (41). Cells were maintained as monolayer cultures in
162-cm2 tissue culture flasks (Costar, Cambridge, MA) in
Dulbecco's modified Eagle's medium containing 10% heat-inactivated
horse serum (Life Technologies, Inc.), 5% fetal calf serum (Life
Technologies) and 1% penicillin-streptomycin solution (complete
medium) in a 5% CO2 humidified atmosphere. Cells were
subcultured once a week by shaking the flask and replating in a 1:4 to
1:6 ratio. The medium was changed every 3 days. The human kidney 293 cells expressing the adenovirus large T antigen (293T) (42) were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum and 1% penicillin-streptomycin. Cells were
subcultured once a week by trypsinization and replating in a 1:3 ratio.
Construction of the Dominant Negative STAT Constructs--
A
FLAG epitope tag was introduced at the 5' end of the full-length rat
Stat1 and Stat3 cDNAs (43) (kindly provided by Dr. G. H. Fey,
University of Erlangen-Nurnberg, Federal Republic of Germany) by
polymerase chain reaction. FLAG-rSTAT1 (FS1) or FLAG-rSTAT3 (FS3)
cloned into the EcoRI and BamHI sites of the
pCMV-1 expression vector was used as template for
oligonucleotide-directed mutagenesis (44). Oligonucleotides used for
the relevant mutations were as follows: FLAG-rStat3-S727A (FS1-SA),
5'-TGCGGGGGGCCATGGCAG-3'; FLAG-rStat3-Y705F (FS1-YF),
5'-GTCTTCAGGAAAGGGGCAG-3'; FLAG-rStat1-S727A (FS1-SA),
5'-CCTCTGGAGCCATGGGAAG-3'; FLAG-rStat1-Y701F (FS1-YF), 5'-GTCTTGATGAATCCAGTTC-3', respectively. The double mutants (FS3-DM and
FS1-DM) contained both Tyr 
Phe and Ser Ala mutations. Each
mutation generated within FS1 and FS3 was verified by double-stranded DNA sequencing using Sequenase (Amersham Pharmacia Biotech) with gene-specific primers. To serve as a control construct for the immunocytochemistry experiment, protein-disulfide isomerase (PDI) (45)
was tagged at the 5' end with FLAG epitope (FPDI) and cloned into the
EcoRI and BamHI sites of pCMV-1 vector.
To verify the expression and the specificity of the wild type and
mutant STAT proteins, 10 µg each of the STAT cDNA construct and
the rat IL-6R (ATCC 63082, PRIL6RC.6; cloned into pCMV1) were
transiently transfected into 293T cells grown on 100-mm dishes using
the calcium phosphate coprecipitation method (46). A preliminary immunoblotting experiment suggested that 293T cells do not express a
detectable level of IL-6R. Cells were incubated with the
precipitates for 12 h and continued in culture in fresh complete
medium overnight. Cells were then stimulated with human recombinant
IL-6 (20 ng/ml; a generous gift from Amgen, Inc., Thousand Oaks, CA)
for 15 min.
Immunoprecipitation and Immunoblot Analyses--
Preparation of
293T cell lysates, immunoprecipitation of FLAG-tagged proteins with M5
monoclonal antibodies, and immunoblot analyses were performed
essentially as described previously (32). To determine the effect of
expression of dominant negative RAS on protein tyrosine phosphorylation
of ERK, cells were grown in complete medium for 1 day and changed to
low serum media overnight before stimulation with NGF (100 ng/ml) for 5 min or IL-6 (30 ng/ml) for 15 min. For the effect of MAPKK inhibitor on
protein tyrosine phosphorylation of ERK, PC12-E2 cells were pretreated with PD98059 (a gift from Parke-Davis, Ann Arbor, MI) for 1 h before stimulation with NGF or IL-6 as described above. The following antibodies were used in this study: mouse monoclonal anti-FLAG antibody
(M5; Eastman Kodak Co.); mouse monoclonal anti-phosphotyrosine (4G10;
Upstate Biotechnology, Inc., Lake Placid, NY); anti-pan-ERK monoclonal
antibody (Transduction Laboratories, Lexington, KY); rabbit polyclonal
anti-GST antibody and anti-Stat3 polyclonal antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA); Anti-active MAPKTM
polyclonal antibody (Promega, Madison, WI); rabbit polyclonal anti-Phospho-Stat3(Ser727) antibody (New England Biolabs,
Inc., Beverly, MA); and rat monoclonal anti-p21ras (259)
antibody (Oncogene Science, Cambridge, MA).
Immunocytochemistry--
PC12-E2 cells were grown on rat tail
collagen-coated glass coverslips in six-well plates and transfected
with 6 µg of DNA using Lipofectin reagent (Life Technologies)
according to the manufacturer's instructions. After incubation for
5 h in Opti-MEM (Life Technologies), cells were allowed to recover
for 24 h in complete medium and then treated with IL-6 (30 ng/ml)
or NGF (100 ng/ml) in Dulbecco's modified Eagle's medium containing
1% horse serum (low serum medium) for 40 h.
Immunostaining of PC12-E2 cells following growth factor stimulation was
performed essentially as described previously (47). For cells
transfected with FLAG-tagged constructs, cells were incubated with
anti-FLAG monoclonal antibody (M5) overnight at 4 °C followed by
rabbit anti-mouse IgG (Jackson Laboratory, Bar Harbor, ME) for 2 h
and fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit IgG
(Molecular Probes, Inc., Eugene, OR) for 2 h at room temperature.
Immunostained cells were identified under an epifluorescence-equipped
microscope, and responsive cells were scored as cells extending
neurites at least two cell bodies in length.
Electrophoretic Mobility Shift Assay (EMSA)--
Preparation of
crude nuclear extracts and EMSA were performed as described previously
(32).
Luciferase Reporter Gene Assays--
The Stat1 reporter gene
construct, pTAL-LucGAS, was constructed by inserting four copies of an
annealed oligonucleotide corresponding to the GAS element from the
murine LY-6E gene (48) into the KpnI and
XmaI sites upstream of the thymidine kinase minimal promoter of the luciferase reporter vector pTAL-Luc
(CLONTECH, Palo Alto, CA). The Stat3 reporter gene
construct pLucTKS3, containing seven copies of the human C-reactive
protein gene acute-phase responsive element, was a generous gift from
Dr. Jove (H. Lee Moffitt Cancer Center and Research Institute, Tampa
Bay, Florida) (49). PC12-E2 cells were grown on rat tail
collagen-coated six-well plates and transfected with a mixture of
plasmids containing STAT reporter constructs (4 µg), pRL-null, a
Renilla luciferase expression plasmid (1 µg; Promega), and
dominant negative STAT constructs (Stat3 constructs, 4 µg; Stat1
constructs, 2 µg) using Lipofectin reagent as described above. After
48 h, cells were stimulated with or without IL-6 (30 ng/ml) or rat
interferon 
(10 ng/ml; Life Technologies) for 6 h, lysed, and
assayed for luciferase activities using the Dual Luciferase Reporter
(DLRTM) Assay System according to the manufacturer's
instructions (Promega). Luciferase activities were normalized to the
internal control Renilla luciferase activity.
Stable Cell Line Expressing Dominant Negative ras--
Dominant
negative ras cDNA (v-Ha-ras-Asn17) was subcloned into
pCMV-Hygro expression vector (kindly provided by Dr. Eric Stanbridge, University of California, Irvine) and used to transfect PC12-E2 cells
using Lipofectin reagent to generate stable cell lines. One day
following transfection, hygromycin (Roche Molecular Biochemicals) was
added to the culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% horse serum), and selection was carried out for 3 weeks until individual colonies could be selected
and screened for p21ras expression. Using immunoblotting
analysis, two clones (DN-RAS12 and DN-RAS27) with high expression level
and one clone with mock transfection (Hygro-CMV) were selected for
further studies.
Neurite Outgrowth--
The neurite outgrowth assay was performed
as described previously (32). For the induction of neurite outgrowth in
cells expressing dominant negative RAS proteins, cells were grown in complete medium for 6 h and changed to low serum medium with the addition of IL-6 (30 ng/ml) or NGF (100 ng/ml) for 1-4 days. For the
effect of MAPKK inhibitor PD98059 on neurite outgrowth, PC12-E2 cells
were grown in complete medium for 16 h, changed to low serum medium, and pretreated with PD98059 (0 to 30 µM) for
1 h before the addition of IL-6, NGF, or no factors (control) for
24 h. The percentage of responsive cells was scored.
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RESULTS |
Construction of Dominant Negative Forms of STAT
Mutants--
Results from previous studies have suggested that IL-6
stimulates neurite proliferation in PC12-E2 cells by a mechanism that is substantially independent of RAS/ERK and that the response is most
likely mediated by activation of Jak/STAT pathways (32). However, Ihara
et al. (35) found STAT activation to be inhibitory of PC12
cell differentiation. To better resolve the role of Jak/STAT pathways
in IL-6-induced neurite proliferation, various dominant negative
mutants of Stat1 and Stat3 proteins were introduced into PC12-E2 cells,
and their effects were tested.
The properties and effectiveness of several dominant negative forms of
Stat1 and Stat3 have been previously characterized (50-53). In the
present study, these inhibitors were prepared by mutating a single
tyrosine residue to phenylalanine (YF), a single serine residue to
alanine (SA), or a combination of both (DM), using
oligonucleotide-directed mutagenesis. The cDNA constructs generated, including wild type rat Stat3 (FS3) and rat Stat1 (FS1) and
dominant negative forms (FS3-YF, FS3-SA, FS3-DM, FS1-YF, FS1-SA, and
FS1-DM), were tagged at the 5' end with a FLAG epitope. Expression and
integrity of the wild type and mutated STATs were first verified by
transiently transfecting each construct into human embryonic kidney
293T cells co-expressing rat IL-6R (Fig.
1). Anti-Stat3 and anti-Stat1 immunoblots
of anti-FLAG immunoprecipitates from 293T cell lysates suggest that all
constructs were expressed robustly and at a similar level in 293T
cells. Anti-phosphotyrosine and anti-phospho-Stat3(Ser727)
blots show that stimulation with IL-6 results in tyrosine
phosphorylation of FS3 and an increased serine phosphorylation (Fig.
1A). It is also noted that serine phosphorylation of Stat3
proteins results in a reduced mobility of FS3 as compared with FS3-SA
shown on the phosphotyrosine blot. When FS3 protein, in which
Tyr705 is replaced by phenylalanine (FS3-YF or FS3-DM) was
expressed, no tyrosine phosphorylation was detected after IL-6
stimulation. When Ser727 on FS3 protein was replaced by an
alanine residue (FS3-SA or FS3-DM), no serine phosphorylation was
detected in the presence or absence of IL-6 stimulation. Similarly, FS1
was phosphorylated on tyrosine after IL-6 stimulation (Fig.
1B). However, no tyrosine phosphorylation was detected in
FS1 containing a phenylalanine at position 701 (FS1-YF or FS1-DM).
Mutation of Ser727 to alanine was confirmed by
double-stranded DNA sequencing.
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Fig. 1.
Expression of STAT constructs and protein
tyrosine and serine phosphorylation of STAT mutants in IL-6 stimulated
293T cells. FLAG epitope-tagged Stat3 (A) and Stat1
(B) cDNA constructs cloned into the pCMV-1 expression
vector were transiently co-transfected with or without rat IL-6R in
293T cells by the calcium phosphate co-precipitation method. Cells were
grown in complete medium for 1 day and continued in culture in low
serum medium for 1 day before stimulation with or without IL-6 (30 ng/ml) for 15 min. Immunoprecipitates of FLAG-tagged proteins from
transfected cells were separated by SDS-polyacrylamide gel
electrophoresis, immunoblotted with anti-phosphotyrosine antibody, and
detected by ECL as shown on the upper panels. In
A, the phosphotyrosine blot was stripped and reprobed with
anti-phosphoserine Stat3(Ser727) antibody, which was again
stripped and reprobed with anti-Stat3 antibody to reveal the FS3
protein level as shown in the bottom panel. In
B, the phosphotyrosine blot was stripped and reprobed with
anti-Stat1 antibody to reveal the FS1 protein level as shown in the
bottom panel.
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The Effects of Dominant Negative STATs on IL-6-induced Neurite
Outgrowth in PC12-E2 Cells--
To determine if neurite outgrowth
induced by IL-6 is mediated by activation of the Jak/STAT pathway,
dominant negative STAT constructs were transiently introduced into
PC12-E2 cells. Cells expressing recombinant proteins were identified by
immunostaining with anti-FLAG monoclonal antibody, rabbit anti-mouse
IgG, and FITC-conjugated goat anti-rabbit antibody and examined under
an epifluorescent microscope (Fig. 2).
IL-6 and NGF induced 72.2 ± 1.1 and 78.6 ± 2.2% responsive
cells in neurite outgrowth assay, respectively, in PC12-E2 cells
transfected with the control construct, FPDI (Fig.
3). Overexpressing wild type FS3 does not
significantly affect neurite outgrowth induced by either NGF or IL-6.
In contrast, neurite outgrowth induced by IL-6 is greatly inhibited in
cells overexpressing FS3-YF, but there is no effect on NGF-induced
neurite outgrowth. The FS3-SA mutant can be phosphorylated on
Tyr705, forming homodimers or dimers with endogenous STATs,
and translocated to the nucleus. Although they may bind DNA, the
transcriptional efficiency is apparently less than the homodimers of
the wild type Stat3. Expression of FS3-SA produces a weak inhibitory
effect on IL-6-induced neurite formation (reduced by about 30%), but, as with FS3-YF, NGF-induced neurite outgrowth is not affected. Expression of FS3-DM produces complete inhibition of the IL-6-induced neurite proliferation. Responsive cells were reduced to control level.
Again, NGF-induced neurite outgrowth was not affected by the expression
of FS3-DM. These results clearly suggest that the activation of Stat3
pathway is essential for IL-6 to induce morphological differentiation
of PC12-E2 cells.
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Fig. 2.
NGF and IL-6-induced neurite outgrowth in
PC12-E2 cells overexpressing wild type and dominant negative Stat1 and
Stat3. PC12-E2 cells plated on collagen-coated glass coverslips
were transiently transfected with the control (FPDI), wild type, and
double mutated forms of Stat3 (FS3 and FS3-DM) or with Stat1 (FS1 and
FS1-DM) cloned into the pCMV-1 expression vector using Lipofectin
reagent. Cells were treated for 40 h with no factors (control), 30 ng/ml IL-6, or 100 ng/ml NGF and immunostained with anti-FLAG
monoclonal antibody, rabbit anti-mouse IgG, and FITC-conjugated goat
anti-rabbit IgG. Cells overexpressing STAT proteins were identified by
epifluorescent microscopy. Bar, 50 µm. The field shown was
selected to demonstrate the STAT expression at high resolution and is
not a statistically significant sample. The values in Fig. 3 are
averages of duplicate samples from 3-5 independent experiments and
were obtained from much broader fields.
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Fig. 3.
The effect of expression of wild type and
dominant negative Stat3 (A), Stat1
(B), and SHC (C) proteins on NGF and
IL-6-induced neurite outgrowth in PC12-E2 cells. Experimental
conditions were the same as described in Fig. 2, except cells
transfected with GST-tagged constructs (C) were
immunostained with anti-GST monoclonal antibody. Cells were treated
with no factors (open column), IL-6
(solid column), or NGF (hatched
column). Responsive cells were identified and scored under
an epifluorescent microscope. Vertical bars
represent S.E. (n = 3-5).
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Although IL-6 predominantly activates Stat3 in PC12-E2 cells, it
transiently activates Stat1 as well (32). To examine if Stat1 plays a
role in IL-6-induced neurite outgrowth in these cells, similar
experiments using wild type and dominant negative mutants of Stat1 were
performed. Interestingly, overexpression of the wild type FS1 protein
inhibits IL-6-induced neurite outgrowth by 60% (Figs. 2 and 3). Mutant
Stat1 proteins also produce partial inhibition of the IL-6-induced
neurite outgrowth. As with wild type and the mutant Stat3 proteins,
Stat1 and the dominant negative Stat1 mutants have no effect on
NGF-induced neurite outgrowth. It appears that the wild type Stat1
proteins exhibit partial inhibitory effects on IL-6 activated Stat3
signaling. It was previously reported that the major STAT-DNA binding
complexes formed in PC12 cells after IL-6 stimulation consist of Stat3
homodimers. Thus, it is likely that overexpressed FS1 proteins alter
the pattern of DNA binding complexes in favor of the formation of Stat1
homodimers or Stat1-Stat3 heterodimers.
The Effect of Dominant Negative STATs on IL-6-induced DNA Binding
Activity--
To determine the effect of dominant negative STATs on
IL-6-induced sis-inducible element (SIE) binding activity,
293T cells were transiently transfected with pCMV-IL-6R together
with expression vectors for the various STAT constructs. EMSAs using
32P-labeled high affinity SIE elements and nuclear extracts
from cells treated with IL-6 were performed. Treatment of cells
expressing FPDI with IL-6 for 15 min leads to formation of three
STAT-DNA complexes, SIFA, -B, and -C (Fig.
4) (54). The slowest migrating complex,
SIFA, is the major complex containing Stat3 homodimers (32). SIFB,
which contains the heterodimers of Stat1 and Stat3, is less intense.
The induction of SIFC, which contains the homodimers of Stat1, is
barely detectable. The pattern of DNA binding complexes is altered in
cells overexpressing FS1. In these cells, the strongest complex formed
after 15 min of IL-6 stimulation is the SIFC. The SIFB complex is not
as intense as SIFC; however, it is stronger than SIFB observed in cells
overexpressing the control, FPDI. The formation of SIFA is inhibited in
these cells. The dominant negative mutant of Stat1, FS1-DM, cannot be
activated by IL-6 to induce SIE binding activities; however, the
formation of native SIFA and SIFB complexes observed in cells
transfected with control construct is modestly reduced (Fig. 4). These
results suggest that overexpression of either wild type FS1 or the
dominant negative FS1-DM inhibits to some degree IL-6-activated Stat3
DNA binding activities.
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Fig. 4.
Stimulation of DNA binding activity by IL-6
in 293T cells overexpressing wild type and dominant negative Stat1 and
Stat3. 293T cells were transiently transfected with
pCMV-1-IL-6R and various STAT constructs as described in the legend
to Fig. 1. The autoradiograph of EMSA is shown in A. Nuclear
extracts were prepared from transfected cells treated with or without
IL-6 (30 ng/ml) for 15 min. EMSA was performed with a
32P-labeled SIE probe. The three protein-DNA complexes
formed (SIFA, SIFB, and SIFC) were separated from the free probes by
electrophoresis on a 4% polyacrylamide gel containing 0.5 × TBE.
The expression level of each protein is shown in B. Total
cellular proteins were prepared from the same transfected 293T cells,
separated by SDS-polyacrylamide gel electrophoresis, and immunoblotted
with the anti-FLAG antibody (M5).
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In cells overexpressing wild type Stat3, the migration of the SIFA
complex containing FS3 homodimers is slightly faster, and the intensity
of this complex is modestly increased as compared with native SIFA
complexes. The FS3-DM is a potent dominant negative inhibitor for
IL-6-activated Stat3 signaling. Treatment of cells expressing FS3-DM
with IL-6 does not stimulate any DNA binding activity. These results
support the view that Stat3 homodimers are most likely the entities
required for the induction of differentiation-related genes by IL-6 and
that a Stat3 but not a Stat1 signaling pathway is preferentially
utilized by IL-6 to induce neurite proliferation in E2 cells.
The Effects of Dominant Negative STATs on IL-6-induced
Transcriptional Activities--
The luciferase reporter gene assays
were performed to further substantiate the transcriptional efficiencies
of dominant negative STATs in PC12-E2 cells. IL-6 does not induce
luciferase activity in cells transiently transfected with control
constructs pLucTK or pTAL-Luc. However, the luciferase activity was
increased by 2.8-fold in cells transiently transfected with the Stat3
reporter plasmid pLucTKS3, containing seven copies of Stat3-specific
binding sites, from the human C-reactive protein gene acute-phase
responsive element (49) (Fig.
5A). Co-expressing wild type
FS3 modestly enhanced IL-6-induced transcription. In contrast,
luciferase activity induced by IL-6 was reduced to basal level in cells
co-expressing FS3-YF or FS3-DM. Expression of FS3-SA also modestly
reduced IL-6-induced luciferase activity, which is consistent with its
inhibitory effect on IL-6-induced neurite outgrowth. These data
confirmed that inhibition of neurite outgrowth by Stat3 mutants indeed
results from inhibition of Stat3-dependent
transcription.
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Fig. 5.
Transcriptional activation by IL-6
(A) and interferon (B) in PC12-E2 cells overexpressing wild type
and dominant negative Stat1 and Stat3. PC12-E2 cells were
transiently co-transfected with a STAT reporter gene construct
(pLucTKS3 or pTAL-LucGAS), an internal control plasmid (pRL-null), and
various STAT constructs using Lipofectin reagent. To serve as controls,
a control reporter gene vector (pLucTK or pTAL-Luc), was used instead
of the reporter gene construct. Forty-eight h after transfection, cells
were stimulated with or without IL-6 (A) or interferon (B) for 6 h. Transcriptional activation is expressed as
fold increase in luciferase activity by IL-6 or interferon over
nonstimulated transfected cells. Vertical bars
represent S.E. (n = 4).
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The results described above suggest that expression of wild type FS1
may inhibit IL-6-activated transcription. However, most STAT proteins
have similar DNA binding preferences; therefore, this possibility
cannot be effectively tested using the artificial reporter plasmid,
pLucTKS3. Since we have reported that IL-6 mildly activates Stat1 in
PC12 cells (32), it is not surprising to observe a 2-fold activation of
luciferase activity by IL-6 in cells co-expressing FS1 (data not
shown). Transcriptional specificity of Stat1 and Stat3 may be
determined by cooperative binding interactions between STAT dimers and
natural sequences of physiologically relevant genes (55). Stat1 may
produce a weaker transcriptional activation than Stat3 on these
sequences, and thus overexpression of Stat1 produces overall diminished
transcriptional and neurite outgrowth responses to IL-6. Since the
genes involved in the neurite outgrowth response are not yet
identified, it is difficult to assess this possibility directly.
In order to further determine the specificity of Stat1 constructs, E2
cells were transiently transfected with Stat1 reporter genes,
pTAL-LucGAS, containing four copies of Stat1-specific binding sites,
from the murine LY-6E gene GAS element (48), together with
various Stat1 constructs. Importantly, interferon , a potent activator of Stat1, induced a much stronger activation of luciferase activity in cells co-expressing FS1 than in cells co-expressing a
control plasmid FPDI (Fig. 5B). In contrast, expression of
FS1-YF or FS1-DM inhibited the luciferase activation by interferon . Unlike the Ser Ala mutation on FS3, expression of FS1-SA produces similar effects as wild type FS1, suggesting that serine
phosphorylation is not essential in the transcriptional activation by
Stat1 in E2 cells.
The Effects of Dominant Negative RAS--
IL-6-mediated
differentiation of PC12-E2 cells may require the integration of other
signals with the Jak/STAT pathway. It has been shown that IL-6-mediated
biological responses and the maximal transcriptional activity of Stat1
and Stat3 require both tyrosine and serine phosphorylation of Stat3
proteins (56). In vitro studies suggest that Stat3 is a
potential substrate for ERK (57, 58), which is transiently and weakly
activated by IL-6 in PC12 cells. Thus, it is possible that the weak
activation of a RAS/ERK pathway by IL-6 contributes to some degree to
the response of IL-6. To test this possibility, PC12-E2 cell lines stably expressing dominant negative RAS (pCMV-Hygro-v-Ha-ras-Asn17) were established. Two clones expressing a high level of dominant negative-p21ras proteins (DN-RAS27 and DN-RAS12) and one clone
mock-transfected with pCMV-Hygro vector, were selected for further
study (Fig. 6). As expected, activation
of ERK kinase (as suggested by phosphorylation on both tyrosine and
threonine residues) by NGF was markedly inhibited by expression of
dominant negative RAS in both DN-RAS27 and DN-RAS12. As shown
previously, in CMV-Hygro cells IL-6 weakly stimulates ERK activity,
which is not detectable in both DN-RAS27 and DN-RAS12 cells. Consistent
with reports by others (59), the dominant negative RAS effectively
abolished the neurite outgrowth induced by NGF in parental PC12 cells
(data not shown), whereas in PC12-E2 cells expressing dominant negative
RAS, the NGF-induced response is reduced by about 40-50% (Fig.
6).
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Fig. 6.
The effect of expression of dominant negative
RAS on protein tyrosine phosphorylation of ERK and neurite outgrowth
induced by NGF and IL-6. PC12-E2 cells were stably transfected
with 10 µg of pCMV-Hygro expression vector containing the dominant
negative RAS-Asn17 cDNA using Lipofectin reagent. Two clones with
high expression levels (DN-RAS12 and DN-RAS27) as well as one clone
with mock transfection (pCMV-Hygro) were selected. A, cell
lysates prepared from cells stimulated with no factors (C),
100 ng/ml NGF for 5 min (N), or 30 ng/ml IL-6 for 15 min
(I) were separated by SDS-polyacrylamide gel
electrophoresis. The lower portion of the blot
was immunoblotted with anti-p21ras antibody (259) as shown in
the upper panel, and the upper portion of the
blot was immunoblotted with anti-active MAPKTM as shown in
the middle panel. The phospho-MAPK blot was
stripped and reprobed with anti-ERK antibody as shown in the
bottom panel. All blots were detected by ECL.
B, to determine the effect of dominant negative RAS on
neurite outgrowth induced by NGF or IL-6, cells were grown in complete
medium for 16 h and changed to low serum medium in the presence of
30 ng/ml IL-6 (solid circle) or 100 ng/ml NGF
(solid triangle) or in the absence of factors
(open circle). The percentage of responsive cells
was scored everyday for up to 4 days. Vertical
bars represent S.E. (n = 3).
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The ability of IL-6 to induce neurite outgrowth in DN-RAS27 and
DN-RAS12 was also examined. The dominant negative RAS produces little
effect on IL-6-induced response. Neurite outgrowth by IL-6 is slightly
reduced after 1 day of treatment in both DN-RAS27 and DN-RAS12 as
compared with pCMV-Hygro-transfected cells. The maximal response by
IL-6 in DN-RAS27 after 4 days of treatment was similar to that observed
in control cells, whereas the response in DN-RAS12 is slightly reduced
by 15%. These results provide direct evidence that a RAS-independent
signaling mechanism is capable of inducing morphological
differentiation by PC12-E2 cells.
The Effect of the Dominant Negative Form of SHC--
Activation of
TrkA by NGF leads to association of SHC through its
phosphotyrosine-binding domain and formation of SHC-Grb2-Sos complexes,
which in turn leads to activation of the RAS/ERK pathway (47). The role
of SHC in IL-6-mediated response is less clear; however, it has been
suggested that SHC may act as an adaptor protein for gp130-mediated
responses (60, 61). SHC associates with phosphorylated gp130 through
its Src homology-2 domain (SH2) domain or with activated Jak2 through
its phosphotyrosine-binding domain. As with activation of TrkA by NGF,
following IL-6 stimulation, formation of SHC-Grb2-Sos complexes may
lead to activation of the RAS/ERK signaling pathway (61). To examine if
SHC-associated signaling pathway plays a role in IL-6-induced neurite
outgrowth, PC12-E2 cells were transiently transfected with dominant
negative forms of SHC, the phosphotyrosine-binding domain of SHC
protein (GST-N-SHC), the SH2 domain of SHC (GST-SH2), or SHC with
triple mutations on tyrosine, GST-SHC-Y239F/Y240F/Y317F (GST-SHC-3YF) (47). It has been shown that overexpression of these GST-SHC mutants in
PC12 cells leads to inhibition of downstream signaling pathways
activated by NGF or EGF. E2 cells expressing control or mutant
constructs were identified by immunostaining using an anti-GST
polyclonal antiserum and FITC-conjugated second antibodies as described
previously. The NGF response was reduced by about 60% at 40 h in
E2 cells expressing GST-N-SHC or GST-SHC-3YF and was not significantly
affected by GST-SH2 (Fig. 3). These results suggest that NGF-induced
neurite outgrowth response is partly dependent on the formation of
SHC-Grb2-Sos1 complexes. In contrast, the response by IL-6 is largely
unaffected by all mutants, suggesting that SHC is not an important
adaptor protein for gp130-mediated signaling mechanisms in PC12-E2 cells.
The Effect of MAPKK Inhibitor PD98059--
RAS targets multiple
effectors and activation of RAS leads to activation of multiple
intracellular signaling cascades (62). Although the RAS/ERK signaling
pathway is necessary for the neuronal differentiation by NGF,
accumulating evidence has suggested that a RAS-dependent
but ERK-independent mechanism may also be involved. A specific MAPKK
inhibitor, PD98059, was used to determine the involvement of ERK in NGF
and IL-6-induced neurite outgrowth. ERK activity was reduced in a
dose-dependent fashion in cells pretreated with PD98059 at
0-30 µM. Although not completely inhibited, ERK activity
was markedly reduced at the maximal concentration tested. PD98059 at
concentrations greater than 30 µM exhibits cytotoxicity
in PC12-E2 cells. The weak activation of ERK by IL-6 was completely
inhibited by treatment with PD98059. It was found that PD98059 at 30 µM reduced the NGF- and IL-6-induced neurite outgrowth
response at 24 h by 20 and 30%, respectively (Fig.
7). These findings suggest that
additional ERK-independent pathways are involved in the NGF response in
E2 cells and that the RAS-ERK pathway is not essential for the
IL-6-mediated response.
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Fig. 7.
The effect of MAPKK inhibitor, PD98059, on
protein tyrosine phosphorylation of ERK and neurite outgrowth induced
by NGF and IL-6 in PC12-E2 cells. A, PC12-E2 cells were
grown in complete medium for 2 days and continued in culture in low
serum medium for 1 day before the treatment. Cells were pretreated with
various concentrations of PD98059 for 1 h before stimulation with
100 ng/ml NGF for 5 min or 30 ng/ml IL-6 for 15 min. Immunoblotting
conditions were the same as described in the legend to Fig.
5A. B, to determine the effect of PD98059 on
neurite outgrowth, cells were grown in complete medium for 16 h,
changed to low serum medium, and pretreated with PD98059 for 1 h
before the addition of 30 ng/ml IL-6 (solid
circle), 100 ng/ml NGF (solid
triangle), or no factors (open circle)
for 24 h. The percentage of responsive cells was scored.
Vertical bars represent S.E. (n = 3).
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DISCUSSION |
Multiple signaling pathways are involved in the regulation of
morphological differentiation by PC12 cells. It is well established that activation of the RAS/ERK pathway is essential for NGF- or bFGF-induced neurite proliferation, although additional signaling pathways may also be needed (63) or, under appropriate conditions, might substitute. In any case, until the essential genes that are
necessary for this process are defined, it will not be possible to
decide a priori which factors will be active in a positive manner and which will be inhibitory.
As with most cytokines acting on various cell types, IL-6 predominantly
activates Jak/STAT pathways in PC12 cells by phosphorylation events.
Tyrosine phosphorylation is required for the dimerization and
translocation of Stat1 and Stat3 to the nucleus; however, the role of
serine phosphorylation is less clear (56-58, 64-66). The present
study demonstrates, using PC12-E2 cells transiently transfected with
dominant negative forms of Stat3 proteins, that the activation of Stat3
is required for the IL-6-induced neurite outgrowth. The induction of
neurite proliferation was markedly inhibited in cells overexpressing
Stat3-YF or Stat3-DM, whose inhibitory functions have been well
characterized (50-53). Stat3 with a Y705F mutation may be recruited to
the phosphorylated tyrosine residues on the cytoplasmic tail of the
gp130 through its Src homology 2 domain, but it cannot be
tyrosine-phosphorylated, dimerized, or translocated to the nucleus upon
IL-6 stimulation. Thus, the dominant negative effect of FS3-YF is most
likely due to its competition with the endogenous Stat3 for docking
sites. When introduced into cultured cells, they efficiently prevent
endogenous Stat3 DNA binding and activation of target gene transcription.
IL-6, in addition to its rapid activation of Stat3 tyrosine
phosphorylation, also stimulates a delayed and transient increase in
serine phosphorylation (32). It has been shown that Stat3 with S727A
mutation may bind DNA but with a reduced transcriptional ability (56).
Overexpression of FS3-SA modestly reduces the neurite proliferation
induced by IL-6. This suggests that full transcriptional activation of
Stat3 in PC12 cells requires both tyrosine and serine phosphorylation,
which is consistent with reports by others that IL-6-mediated response
requires an H7-sensitive kinase (67). The identity of the serine
kinases involved, however, is not clear. Overexpression of wild type
Stat3 did not enhance the response to IL-6, indicating that the
biological response to IL-6 in PC12 cells is most likely limited by the
extent of activation and formation of the receptor and gp130 signaling
complexes. Collectively, these results support the view that
IL-6-induced neurite outgrowth is dependent on activation of the Stat3.
In PC12 and PC12-E2 cells, IL-6 activates both Stat3 and Stat1
proteins. However, It induces a more sustained and stronger stimulation
of tyrosine phosphorylation of Stat3 than Stat1, which correlates with
a more intense formation of SIFA than SIFB and SIFC (32). In addition,
the expression of Stat3 is up-regulated in IL-6-treated cells. If Stat1
and Stat3 exhibit similar biological functions in PC12 cells and a
stronger activation of Stat3 is merely a reflection of the relative
concentration of endogenous Stat3 to Stat1, it would be expected that
overexpression of Stat1 would either enhance or at least have no effect
on the response to IL-6. On the contrary, overexpression of Stat1
molecules leads to a partial inhibition of the neurite outgrowth by
IL-6. It was found that overexpression of FS1 alters the pattern of
STAT-DNA complexes. The inhibition on neurite proliferation correlates with an inhibition of the activation of endogenous Stat3 homodimers or
formation of SIFA. It appears that Stat3 homodimers are the transcription factors involved in the regulation of PC12
differentiation, whereas Stat1 homodimers or Stat1-Stat3 heterodimers
display a negative effect on Stat3-mediated mechanisms. These results
further suggest that Stat1 and Stat3 proteins are not only
differentially regulated in PC12 cells by IL-6, but also they may play
opposite (or at least different) roles in the regulation of neuronal differentiation.
Similar to the dominant negative effect of FS3-YF, the mechanism by
which Stat1 acts as a partial dominant negative inhibitor of Stat3
signaling pathway is apparently through competition with endogenous
Stat3 for the docking sites on the cytoplasmic domain of gp130. It has
been demonstrated that activation of Stat3 and Stat1 via the
cytoplasmic domain of gp130 is mediated by multiple independent docking
sites (68, 69). Four carboxyl-terminal tyrosine-containing motifs
involving Tyr767, Tyr814, Tyr905,
and Tyr915 are able to recruit Stat3. However, only two of
the four tyrosine motifs surrounding Tyr905 and
Tyr915 are capable of activating Stat1. This may partly
explain the preferential activation of Stat3 by IL-6 and the fact that
overexpression of Stat1 produces a partial inhibitory effect on Stat3 activation.
The differential role of Stat1 and Stat3 in cellular regulation has
also been suggested in other cell systems. It has been reported that
activation of Stat3 but not Stat1 is required for IL-6-induced terminal
differentiation of myeloid leukemia M1 cells (51, 52, 70) as well as
EGF receptor-mediated cell growth in transformed squamous epithelial
cells (71). Although Stat1 is critical for interferon signaling and
innate response to viral and bacterial infection, recent studies
suggest that it also regulates caspase expression and promotes
apoptosis (72-74). The specific role of Stat1 in PC12 cells, however,
was not determined in the present study.
Induction of neurite outgrowth by NGF but not EGF has been attributed
to the ability of NGF to induce a sustained activation of ERK (39, 75).
EGF induces a short and relatively weak activation of ERK in both PC12
and PC12-E2 cells and fails to stimulate neurite proliferation in
either. IL-6 is capable of activating multiple signaling pathways
including RAS/ERK in many cell types; however, it has little effect on
ERK in some neuronal cells including both PC12 and PC12-E2 cells (60,
61, 76-79). In fact, the effect on ERK by IL-6 is delayed and much
weaker than that induced by EGF (32, 33). Thus, the activation of ERK
by IL-6 alone would not be expected to produce a signal sufficient to
induce neurite proliferation. Since a synergistic response was observed
between IL-6 and subthreshold concentrations of NGF or EGF, it is
possible that weak activation of the RAS/ERK pathway may contribute in some way to the overall response. Data presented in the present study,
however, do not identify a significant contribution of RAS/ERK pathway
to the induction of neurite proliferation by IL-6. IL-6 is capable of
inducing robust neurite outgrowth in PC12-E2 cells expressing dominant
negative RAS or SHC entities. The MAPKK inhibitor, PD98059, did produce
a slight reduction in the percentage of responsive cells, so the weak
activation of ERK may contribute to cell cycle arrest but is not likely
to be important for the IL-6-induced neurite proliferation.
The results presented herein are in contrast to the findings of Ihara
et al. (35). These workers utilized PC12 cells microinjected with chimeric receptors consisting of the extracellular domain of the
granulocyte colony-stimulating factor receptor and the cytoplasmic
domain of gp130, including various mutations and truncations. The cells
were pretreated with NGF for 1-2 h and then stimulated with
granulocyte colony-stimulating factor to activate the chimera. They
observed that elimination of the STAT binding sites on gp130 actually
stimulated differentiation and concluded that IL-6 also induced neurite
outgrowth via ERK activation (as with NGF). The Stat3 activation was
deemed to be inhibitory, and they proposed that the brief exposure of
the cell to NGF suppressed the Jak/STAT pathway, allowing the ERK
pathway to then be manifested. Because of the nature of the
experimental system (microinjection of single cells), these studies did
not allow any direct evaluation of the pathways stimulated, and the
specificity of the chimeras was dependent on evaluation in other cell
types (70). Under the conditions of their experiments, ERK activation
may indeed become the dominant pathway in PC12 cells (80). However,
clearly Stat3 activation is not inherently inhibitory and can, at least
in PC12-E2 cells, lead directly to differentiation. It should be noted
that Ihara et al. (35) studied native PC12 cells, while the
studies reported herein were done with PC12-E2 cells, and undefined
differences between them may underlie the differences in response observed.
The nature of the PC12 cells used in signaling experiments is also
clearly important. IL-6-induced differentiation of PC12 cells is
dependent on other treatments or conditions than are found in native
PC12 cells (not previously exposed to stimuli). Primed cells or PC12-E2
cells give robust, transcription-dependent responses to
IL-6. More transient exposure to NGF (1-2 h) (35) can also produce a
responsive condition; however, we observed that only one of three PC12
cell lines in our laboratory showed this response (data not shown).
Thus, this "initiation" phenomenon requires certain cellular
conditions (in addition to the NGF-induced effects) to achieve IL-6
responsiveness. We have previously suggested that priming by NGF leads
to cell cycle arrest through the production of one or more suppressor
proteins. These, or similar functional entities, are presumed to
already be expressed in PC12-E2 cells. The response to IL-6 is
dependent on these entities (and IL-6 cannot induce their synthesis
directly). This is supported by the observation that primed cells
remain responsive to IL-6 only transiently (~3 days), while PC12-E2
cells are permanently responsive. These observations clearly indicate
that a pathway other than ERK is required for the full IL-6 response
and is entirely consistent with the findings reported herein that that
pathway is contributed by Stat3 activation.
Regulation of neuronal functions involves multiple signaling pathways.
Activation of either RAS/ERK by polypeptide growth factors such as NGF
or bFGF or activation of Jak/STAT pathway by cytokines such as IL-6 or
ciliary neurotrophic factor leads to trophic, survival, or
differentiative effects in various cultured cells. Thus, activation of
these two pathways may lead to opposing physiological responses (17,
70). The data from the present study suggest that, in an appropriate
cellular environment, stimulation of these two parallel signaling
pathways by a distinct class of extracellular signaling polypeptides
may act collaboratively in the regulation of neuronal differentiation.
§
TOP
ABSTRACT
INTRODUCTION
