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The Journal of Neuroscience, May 15, 1998, 18(10):3620-3629
Multiple Routes to Astrocytic Differentiation in the CNS
Prithi
Rajan and
Ronald D. G.
McKay
Laboratory of Molecular Biology, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
20892-4157
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ABSTRACT |
Ciliary neurotrophic factor (CNTF) acts instructively to switch
multipotent stem cells of the CNS to an astrocytic fate. Here we show
that CNTF causes activation of janus kinase-signal transducers and
activators of transcription and mitogen-activated protein kinase (MAPK)
pathways with differential kinetics in these cells. Inhibition studies
indicate that activation of the MAPK pathway is required early in the
differentiation process, whereas activation of signal transducer and
activator of transcription (STAT) proteins is required for commitment
to an astrocytic fate. Bone morphogenetic proteins have also been shown
to cause astrocytic differentiation but do not cause STAT activation or
astrocytic differentiation in fibroblast growth factor 2-expanded fetal
stem cells used here. These results show that there are two distinct
routes to initiate astrocytic commitment in multipotent CNS
precursors.
Key words:
astrocytic differentiation; CNS stem cells; mammalian
CNS development; intracellular signaling; CNTF; BMP4; EGF
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INTRODUCTION |
Multipotent cells that give rise to
neurons, astrocytes, and oligodendrocytes have been defined using both
monolayer and aggregate culture systems. We have used stem cells
isolated from rat embryos to study the molecular correlates of
differentiation leading to the development of astrocytes. When
maintained in a proliferative state in fibroblast growth factor (FGF),
these cells remain multipotent and can differentiate into the three
major cell types of the CNS: neurons, astrocytes, and oligodendrocytes.
Ciliary neurotrophic factor (CNTF) causes 98% of these cells to
differentiate into astrocytes, as specified by glial fibrillary acidic
protein (GFAP) expression (Johe et al., 1996 ). Epidermal growth factor
(EGF) also promotes astrocytic differentiation as EGF-expanded CNS stem cells yield a higher number of astrocytes on mitogen withdrawal (Johe
et al., 1996). Bone morphogenetic proteins (BMPs) have been shown to
induce astrocytic differentiation in cells that are isolated from the
fetal subventricular zone and grown as aggregates in the presence of
EGF (Gross et al., 1996). Both CNTF and BMP are thought to act
instructively on multipotent cells, committing them to an astrocytic
fate. The robust response to CNTF provides a technically accessible
experimental paradigm for a biochemical dissection of the mechanism of
lineage determination by CNTF.
CNTF signaling is accomplished through several pathways. The
best-defined are the signal cascades that involve the janus kinases (JAKs) and the mitogen-activated protein kinase (MAPK) pathway. Signaling is initiated by members of the JAK family of tyrosine kinases, specifically Jak1, Jak2, and Tyk2 (Lutticken et al., 1994;
Stahl et al., 1994). On activation, Src homology 2 (SH2) domain
proteins bind to the receptor. These proteins include the signal
transducers and activators of transcription (STAT) proteins (Stahl et
al., 1995), activation of which leads to direct transcriptional activation (Symes et al., 1994). Activated Jak2 can also cause the
activation of MAPK possibly through a ras-raf pathway that activates
transcription factors including cAMP response element-binding protein
(CREB) and TCF/Elk (Winston and Hunter, 1996). STATs have also been
shown to be activated by EGF (Fu and Zhang, 1993; Schindler and
Darnell, 1995), and this activation could be involved in astrocytic differentiation. In contrast, BMPs signal by activating Sma-MAD homolog
(SMAD) proteins, a distinct family of transcriptional regulators (Lagna
et al., 1996; Liu et al., 1996).
Effects of CNTF are mirrored by leukemia inhibitory factor (LIF) in
CNTF-responsive cells, because the signaling moieties of the two
receptors are identical. Mice lacking LIF receptor  (LIFR-) show
a phenotype of decreased numbers of astrocytes (Ware et al., 1995). In
this report we show that activation of both the MAPK and JAK-STAT
pathways are positively coupled to astrocytic differentiation in
vitro. Inhibition of activation of the MAPK pathway delays the
initiation of differentiation, whereas inhibiting Stat3 function causes
a total block in astrocytic differentiation. However, there is no
activation of the JAK-STAT system by either EGF or BMP4 in the cell
system studied here. These results show that distinct molecular
mechanisms initiate astrocytic commitment in response to BMP4 and CNTF
in multipotent CNS precursors.
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MATERIALS AND METHODS |
Materials
DMEM/F-12 was obtained from Life Technologies (Gaithersburg,
MD). Insulin, transferrin, and fibronectin were from Intergen (Purchase, NY), and progesterone, selenium, putrescine, polyornithine, 1,4-diazabicyclo-(2.2.2)-octane (DABCO), Tween 20, bovine serum albumin, and dimethyl sulfoxide (DMSO) were from Sigma (St. Louis, MO).
Horseradish peroxidase-tagged secondary antibodies for enhanced chemiluminescence (ECL) and poly(dI-dC) were from Boehringer Mannheim (Indianapolis, IN). CNTF, EGF, and FGF were obtained from R & D Systems
(Minneapolis, MN), and BMP4 was from Genetics Institute (Cambridge,
MA). PD098059 was obtained from Research Biochemicals (Natick, MA). The
ECL kit was obtained from Pierce (Rockford, IL). The following were the
antibodies used in this study: GFAP monoclonal from ICN (Irvine, CA),
GFAP polyclonal from Chemicon (Temecula, CA), antibody against the FLAG
epitope from Eastman Kodak (Rochester, NY), Stat1 monoclonal  -ISGF3
(anti-ISGF3) and -pan-ERK antibody from Transduction Laboratories
(Lexington, KY), and Stat3 polyclonal from Santa Cruz Biotechnology
(Santa Cruz, CA). The appropriate fluorescence-tagged secondary
antibodies for immunofluorescence were obtained from Jackson
ImmunoResearch (West Grove, PA).
Culture of neuroepithelial stem cells and
differentiation protocol
Cultures of stem cells were prepared according to the protocol
of Johe et al. (1996). Briefly, cerebral cortices were dissected from
embryonic day 14 (E14) rat embryos; cells were mechanically dissociated
by trituration and were plated on dishes coated with 15 mg/ml
polyornithine and 1 mg/ml fibronectin at a concentration of 1 million
per 10 cm dish. Cells were maintained in 10 ng/ml FGF. Cultures were
passaged on approximately the fourth day. Experiments were performed on
cultures either in the first or second passage.
Cultures were differentiated into astrocytes by treating with 10 ng/ml CNTF.
Electrophoretic mobility shift assay
The preparation of nuclear extracts from treated and untreated
cells was prepared according to the protocol of Symes et al. (1994).
All treatments were performed at 10 ng/ml except for EGF, which was
performed at 20 ng/ml. Cells were treated with CNTF every 24 hr for
time points that were >1 d. Electrophoretic mobility shift assay
(EMSA) was also performed according to the protocol of Symes et al.
(1994). The sequence of the SIE probe used was 5'-AGCTTCATTTCCCGTAAATCCCTA-3' and 3'-AGTAAAGGGCATTTAGGGATTGA-5'. For
supershift assays, the relevant antibody was included in the binding
reaction.
Immunofluorescence
Double-immunofluorescent staining of FLAG-Stat3 and
GFAP. Cells were fixed with freshly mixed methanol and acetone in
a 1:1 ratio for 2 min at room temperature. They were washed four times in Tris-buffered saline (TBS) and calcium (0.05 M Tris HCl,
pH 7.4, 0.15 M NaCl, and 1 mM
CaCl2). Double immunofluorescence was performed with
a biotinylated anti-FLAG monoclonal antibody at 30 µg/ml and
anti-GFAP polyclonal antibody at a 100-fold dilution. Primary
antibodies were left on the cells for 1 hr at room temperature or
overnight at 4°C. The cells were then washed with TBS and calcium for
1 hr with four changes and incubated with streptavidin-FITC- and
lissamine rhodamine-coupled goat anti-rabbit antibody (100-fold dilution) for 30 min to 1 hr at room temperature. After four more washes cells were mounted in 70% glycerol in PBS with 2% DABCO.
GFAP immunofluorescent staining. Cells were fixed with 4%
paraformaldehyde in PBS for 15 min at room temperature. After three washes in PBS they were incubated in blocking solution (PBS with 0.1%
Triton X-100 and 5% normal goat serum) for 1 hr. Cells were then
incubated with monoclonal GFAP antibody at a 200-fold dilution for 2 hr
at room temperature. After four washes in PBS with 0.1% Triton X-100
(PBST), cells were incubated with a 100-fold diluted solution of
FITC-coupled donkey anti-mouse antibody for 1 hr at room temperature.
After four more washes in PBST and one wash in PBS, cells were mounted
in 70% glycerol and 2% DABCO. Dilution of all antibodies was
performed in the blocking solution.
Nestin immunofluorescent staining. The protocol used was
identical to the GFAP staining. Cells fixed in 4% paraformaldehyde were stained with a nestin antibody at a 500-fold dilution. Appropriate secondary antibodies were used at a 100-fold dilution.
Immunoblotting
Cells were washed once in PBS and lysed in NP-40 lysis buffer
(50 mM Tris HCl, pH 8.0, 170 mM NaCl, and 0.5%
NP-40). After a 20 min incubation, extracts were cleared by
centrifuging at 14,000 rpm in a Microfuge for 20 min. All operations
were performed at 4°C. Extracts were fractionated on 12.5% SDS-PAGE
(acrylamide/bisacrylamide ratio of 30:0.2), and the separated proteins
were transferred onto nitrocellulose filters. After blocking in
TBS-Tween (TBST; 20 mM Tris, pH 7.6, 137 mM
NaCl, and 0.1% Tween 20) with 2% bovine serum albumin, filters were
incubated with primary antibody at a 1000-fold dilution for 3 hr at
room temperature. After three washes with TBST, the appropriate
secondary antibody conjugated to horseradish peroxidase was used at a
20,000-fold dilution, and ECL was performed according to the
manufacturer's instructions. All antibody dilutions were performed in
the blocking solution.
Transfection, infection, and PD098059 experiments
Transfections were performed on cells in first passage by the
calcium phosphate method. The plasmids used were the wild-type (WT)
Stat3 and a DNA-binding mutant with a three-amino acid mutation at
residues 462-464 (VVV AAA). Both plasmids had an N-terminal FLAG and
were described by Horvath et al. (1995). The precipitate was left on
the cells for 2 hr; the cells were washed and left in FGF for 48 hr.
Cells were then treated for 48 hr with 10 ng/ml CNTF.
Infections and PD098059 experiments were performed on cells in first or
second passage. An adenovirus vector expressing a constitutively
activated form of MAPK kinase (MAPKK) was used. The activated form of
the protein has a two-amino acid substitution (S218E and S222E) and its
nuclear localization signal is deleted (deletion 32-51). Cells were
infected at the indicated multiplicity of infection and fixed 24 or 48 hr after infection.
A 20 mM stock of PD098059 was made in DMSO. Cells were
treated at a final concentration of 10 or 20 µM for 24 or
48 hr before fixing in paraformaldehyde. For the 48 hr time points,
cells were retreated with CNTF and PD098059 at 24 hr. The CNTF-treated
cells that serve as control were treated with the relevant amount of DMSO. There is no appreciable difference in differentiation of DMSO-treated cells compared with untreated cells (data not shown).
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RESULTS |
CNTF causes differentiation of stem cells into astrocytes
Stem cells maintained in FGF were homogeneous in morphology and in
their expression of nestin, a marker of neuroepithelial stem cells
(Lendahl et al., 1990). A transient treatment with CNTF is sufficient
to commit stem cells to an astrocytic fate in vitro (Johe et
al., 1996). Exposure to CNTF causes these cells to express GFAP (Fig.
1). Consistent with in vivo
observations, the intensity of nestin staining decreased with an
increase in GFAP expression (Hockfield et al., 1985; Frederiksen et
al., 1988).
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Figure 1.
CNTF causes differentiation of stem cells into
astrocytes. Cultures of FGF-expanded stem cells were prepared as
described previously (Johe et al., 1996) and treated for 2 d with
10 ng/ml CNTF. After fixation cells were stained with a nestin antibody
(a stem cell marker; A, C) and -GFAP antibody (an
astrocytic marker; B, D). Scale bar: D,
100 µm. There is no GFAP expression when cells are maintained in FGF.
These cells are uniformly positive for nestin. GFAP is expressed after
CNTF treatment. Several of the differentiated cells also express
nestin, but nestin is downregulated in cells intensely fluorescent for
GFAP.
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STAT proteins are activated by CNTF in neuroepithelial
stem cells
One of the major cytoplasmic signaling pathways activated by CNTF
is JAK-STAT. Activation of STAT proteins involves phosphorylation of a
specific tyrosine residue (Shuai et al., 1993, 1994). Activated STAT
proteins can be detected by their ability to dimerize and bind specific
DNA target sites. This interaction can be detected by an EMSA. Specific
nucleoprotein complexes were formed when a STAT-specific DNA-binding
sequence (SIE) was incubated with nuclear proteins prepared from stem
cells treated with CNTF. Complexes labeled "a" and "b" were
formed when the cells were treated with CNTF for times ranging from 15 min to 8 d (Fig. 2). The amount of
complex b formed decreased with time, whereas complex a was activated
for prolonged periods. The constitutive complex that migrates just
below complex b is present even in FGF-treated cells and is not induced
by CNTF treatment. Although there is some Stat3 complex in the control
conditions, indicating basal levels of activation of this protein in
the cell, these data clearly show that CNTF rapidly activates proteins
that bind to the SIE probe.
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Figure 2.
STAT proteins are activated by CNTF in
neuroepithelial stem cells. Stem cell cultures were treated with FGF
and CNTF either separately or in combination and nuclear proteins were
prepared from these cells at the time points mentioned. Cells were
treated every 24 hr for time points >1 d. Cells were mechanically
removed from the culture dish in PBS, and nuclear proteins were
prepared. EMSA assays were then performed to determine the extent of
activation of the STAT proteins in these extracts. The complexes formed
were resolved on a 6% nondenaturing polyacrylamide gel with 0.5× TBE
buffer at 4°C. The positions of the complexes formed as a result of
CNTF treatment are marked a and b. There
is prolonged activation of STAT proteins as seen by the continued
appearance of complex a and, to a lesser extent, complex
b.
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The GFAP promoter may be used as a paradigm to study transcriptional
regulation during astrocytic differentiation, because it is robustly
and specifically expressed in these cells. Promoter analysis of the
GFAP promoter in CG4 cells showed that regions  1857 to 1546 and
384 to 106 are important for CNTF-induced GFAP expression (Kahn et
al., 1997). Another report of promoter analysis of the GFAP promoter in
U251 cells showed the following regions to be necessary and sufficient
for GFAP expression: 1757 to 1604, 1612 to 1489, and 132 to
57 (Besnard et al., 1991). On examination of the GFAP promoter we
identified three putative STAT sites: 1512 to 1504, TTCCGAGAA;
1292 to 1284, TTCCCAGAA; and 277 to 285, TTCCTGGAA; one of which
(1512 to 1504) is stronger in STAT complex formation by EMSA than
the other two (data not shown). These observations suggest that GFAP
induction by CNTF may be mediated by direct interaction of STAT
complexes with the GFAP promoter.
The activated STAT proteins are Stat1 and Stat3
Supershifting of nucleoprotein complexes with antibodies against
specific transcription factors indicate the involvement of these
proteins in the formation of that complex. The identity of the STAT
proteins activated by CNTF was established by supershift assays (Fig.
3). Complex b was supershifted by the
Stat1 antibody, forming complex c, indicating that Stat1 is a part of
complex b (Fig. 3a, lane 4). An -Stat3
antibody caused the disappearanceof both complexes a and b, forming the
supershifted complex d (Fig. 3b, lane 8). Thus,
complex a comprises homodimers of Stat3 complexed with DNA, whereas
complex b comprises heterodimers of Stat1 and Stat3 with DNA.
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Figure 3.
The STAT proteins activated are Stat1 and Stat3.
a, Extracts prepared from cells treated with CNTF for 15 min were used here. EMSA was performed as described in the legend to
Figure 2, except for the inclusion of either a relevant antibody or
unlabeled SIE oligomer DNA. a, lanes 3, 4, An antibody against Stat1 (-Stat1) was included in the
incubation during complex formation. The resultant supershifted
complexes are indicated by c. The efficiency of
formation of complex b is simultaneously reduced
(compare with control, lane 2). b,
Similarly, complex formation in lanes 7 and
8 was performed in the presence of Stat3 antibody
(Stat3). A supershifted band d forms,
with a simultaneous reduction in complexes a and
b (compare lanes 6, 8). c,
EMSA was performed with a 20-fold excess of unlabeled SIE oligomer.
There is a decrease in the intensity of complexes a and
b and the constitutive complex formed in the presence of
the competitor DNA.
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The specificity of the complexes formed was determined by competition
with 20-fold excess of unlabeled SIE oligo (Fig. 3c, lanes 11, 12). Both the induced (complexes a and b) and the
constitutive complexes were competed with the unlabeled oligo,
indicating that both are specific. However, the constitutive complex
does not contain either Stat1 or Stat3, as shown by the supershift
assays. There was no competition with an unrelated probe of similar
length (data not shown).
CNTF induces the formation of three complexes in several other systems
studied (Bonni et al., 1993; Symes et al., 1994; Rajan et al., 1995,
1996). The largest is composed of Stat3 homodimers, the middle of Stat1
and Stat3 heterodimers, and the fastest-migrating one of Stat1
homodimers. From these previous observations it is probable that a
third complex comprising homodimers of Stat1 migrates at the same
position as the constitutive complex that forms just below complex b.
It is of interest to note that there are two distinct complexes
supershifted by the Stat1 antibody, indicating that there are two
species of Stat1 activated by CNTF treatment in these cells. These
results show that both Stat1 and Stat3 are activated by CNTF in the
stem cells.
Blocking of Stat3 function prevents astrocytic differentiation
by CNTF
The requirement of STAT activation in CNTF-mediated astrocytic
differentiation was determined by assaying the effect of blocking Stat3
transcriptional function. Stat3 was targeted because EMSA indicated
that it was the major protein that was activated. Plasmids expressing
FLAG-tagged WT and mutant forms of the Stat3 protein (Horvath et al.,
1995) were transfected into cells. The mutant Stat3 has a three-amino
acid substitution in the DNA-binding domain, thus functioning as a
dominant negative because of loss of DNA-binding function. The effect
of the overexpressed transfected proteins on astrocytic differentiation
was determined by double immunofluorescence with antibodies against
GFAP and the FLAG epitope (Fig.
4a). Transfection of WT Stat3
allows astrocytic differentiation in 80% of the cells, whereas
differentiation takes place in only ~25% of cells transfected with
the mutant Stat3 (Fig. 4b), indicating that Stat3 function is required for astrocytic differentiation. The fact that
differentiation is not 100% in the WT transfectants and 0% in the
mutant one could be attributable to the fact that perturbations in this
important signaling pathway cause responses of the cells to be skewed
or, in the case of the mutant transfections, that the levels of
dominant negative protein expressed are not sufficient to override the effect of the endogenous WT protein.
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Figure 4.
Effect of blocking Stat3 function on astrocytic
differentiation. a, Cells were transiently transfected
by the calcium phosphate method with plasmids expressing either
wild-type (WT) or dominant negative mutant of
Stat3 defective for DNA binding (mutant), both proteins
expressing an N-terminal FLAG epitope. Forty-eight hours after
transfection cells were treated with CNTF for 48 hr and fixed with
freshly mixed methanol and acetone in a 1:1 ratio for 2 min at room
temperature, and double-immunofluorescent staining was performed for
FLAG and GFAP. A-C, Representative experiment of cells
transfected with the mutant Stat3. D-F, Representative
experiment of cells transfected with WT Stat3. A, D show
staining specific for the FLAG epitope on the transfected proteins,
B, E for GFAP, and C, F for both
antigens. b, Based on experiments performed as described
in Figure 3a, the percentage of transfected cells
expressing GFAP was plotted for each of the two plasmids. Blocking of
Stat3 function with a dominant negative protein inhibits CNTF-mediated
astrocytic differentiation.
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CNTF treatment causes activation of MAPK in neuroepithelial
stem cells
Activated Jak2 can cause the activation of MAPK, possibly through
a ras-raf pathway (Winston and Hunter, 1996). To determine whether
CNTF activates the MAPK pathway in the stem cells, immunoblots were
performed on cell extracts treated with CNTF for time points ranging
from 15 min to 24 hr. CNTF causes activation of p44 MAPK in the
neuroepithelial stem cells (Fig. 5). A
time course shows that this activation is transient and peaks at 15-30
min. The levels of activation return to baseline by 4 hr after CNTF
treatment (Fig. 5, compare lanes 9, 6).
FGF-treated cells were used as a positive control because FGF is a
known activator of MAPK.
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Figure 5.
CNTF activates MAP kinase in the neuroepithelial
stem cells. NP-40 extracts of cells treated with FGF (lanes
1-5) or CNTF (lanes 6-9) for the mentioned
time points were resolved on 12.5% SDS-PAGE, proteins were blotted on
nitrocellulose, and filters were probed with an anti-pan-ERK antibody.
After incubation with the appropriate horseradish peroxidase, coupled
secondary antibody protein bands were visualized with ECL. The major
protein seen is the p44 MAP kinase, the phosphorylated form moving as
the heavier band at time points in which activation is elicited.
Activation of MAP kinase is slightly prolonged after FGF treatment when
compared with CNTF treatment. Activation of MAP kinase is completely
back to control levels (Mock, lane
6) at 4 hr after CNTF treatment (lane
9).
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Inhibition of activation of MAPKK causes a delay in astrocytic
differentiation by CNTF
To determine the relevance of MAPK activation in astrocytic
differentiation, activation of MAPK was blocked by inhibiting the
activation of its upstream activator, MAPKK. PD098059 is a specific
inhibitor of MAPKK at concentrations of  50 µM (Alessi et al., 1995). The effect of adding PD098059 to cells differentiating in response to CNTF was determined, as shown in Figure
6, a and b.
Addition of CNTF to cells in which activation of MAPKK has been
inhibited leads to a complete lack of differentiation at 24 hr after
the addition of CNTF (Fig. 6a, compare C,
D with B). However, by 48 hr the extent of
differentiation in CNTF-treated cells is comparable both in the
presence and absence of PD098059 (Fig. 6a, compare
K, L with J). Thus there is a
delay in the onset of differentiation by CNTF in the absence of normal
levels of MAPK activation.
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Figure 6.
Blocking MAPK activation causes a delay in
astrocytic differentiation. a, MAPKK inhibitor PD098059
was used to block MAPK activation. Cells were treated with CNTF for 24 or 48 hr in the presence or absence of 10 or 20 µM
PD098059, fixed with 4% paraformaldehyde, and stained for GFAP. For
the 48 hr time points, cells were retreated with CNTF and PD098059 at
24 hr. A representative experiment of the data plotted in
b is shown. A-D, I-L,
Immunofluorescent staining of GFAP. E-H,
M-P, Corresponding bright-field (phase) images of these
fields, E being the same field as A and
so on. A-H, Images of cells fixed 24 hr after treatment
and I-P at 48 hr after treatment. Scale bar:
P, 100 µm. b, Average percentage of
GFAP-positive cells was plotted for each condition.
Squares, 24 hr time points; diamonds, 48 hr time points. Astrocytic differentiation is prevented by PD098059
only at the 24 hr time point.
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The CNTF-mediated activation of MAPK in these cells peaks at 15 min,
whereas the effect of its inhibition is seen at 24 hr, indicating that
activation of MAPK leads to events downstream that regulate
differentiation. The eventual differentiation at 48 hr may be a
consequence of the residual activation of MAPK in the cell despite the
inhibitor (see Fig. 7a), which
is sufficient for differentiation to occur in a delayed manner. These
results indicate that one of the earliest steps in CNTF-mediated
astrocytic differentiation includes the activation of MAPK.
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Figure 7.
PD098059 inhibits MAPK activation but does not
affect STAT activation. a, Cells were treated with CNTF
in the presence or absence of 20 µM PD098059 for the
indicated time points, and activation of MAPK was analyzed as described
in Figure 5. At 20 µM, PD098059 blocks most of the MAPK
activation caused by CNTF (compare lanes 3, 2 and
5, 4). b, Nuclear extracts
prepared from stem cells treated with CNTF for the time points
indicated, in the presence or absence of PD098059. EMSA was performed
on these extracts as described in Figure 2. The specific complexes
formed as a result of CNTF treatment are marked a and
b. There is no inhibition of STAT complex formation seen
at early (1h, lanes 1, 2) or late
(48h, lanes 7, 8) time points in the
presence of 20 µM PD098059.
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Overexpression of constitutively active MAPKK causes transient
GFAP expression
Since the previous set of experiments established that MAPK is
required in the initiation of astrocytic differentiation, we investigated the effects of aberrant activation of MAPKK in the absence
of CNTF treatment. FGF-expanded stem cells were infected with an
adenovirus vector expressing a constitutively activated form of MAPKK.
A representative set of experiments is shown in Table
1. The result is the inverse of that seen
in Figure 6. There was a transient expression of GFAP at 24 hr after
infection that was lost at 48 hr. Thus MAPK activation in the absence
of other CNTF-activated pathways leads to a transient appearance of a
differentiated astrocytic phenotype.
Treatment of stem cells with PD098059 blocks activation of MAPK but
does not affect STAT activation
The activation of MAPK by CNTF was appreciably inhibited by
PD098059, as seen in Figure 7a. The intensity of the heavier
bands denoting activation are reduced in lanes 3 and
5 (+PD098059) when compared with lanes 2 and
4 (control). Because MAPK and STAT activation both lead to
astrocytic differentiation, it is possible that there is an interaction
between the two pathways, and the inhibition of differentiation seen in
Figure 6 could be attributed to a block of STAT activation by PD098059,
via the inhibition of MAPK activity. As detected by EMSA, PD098059 did
not affect STAT activation in response to CNTF (Fig. 7b).
The amounts of complexes a and b formed are unchanged in the presence
and absence of PD098059 at early (1 hr, lanes 1, 2) and late
(48 hr, lanes 7, 8) time points. It appears that there is no
interaction between the MAPK and JAK-STAT pathways at the level of
STAT activation in the FGF-expanded stem cells.
EGF and BMP4 do not activate STAT proteins
EGF treatment of FGF-expanded stem cells leads to astrocytic
differentiation (Johe et al., 1996). In addition, EGF-treated aggregates from the fetal subventricular zone show enhanced astrocytic differentiation in response to BMPs 2, 4, and 7 (Gross et al., 1996).
These observations raise the possibility that EGF and BMPs might
activate the STAT signaling system in CNS stem cells. Treatment of
cells with EGF and BMP4 does not cause activation of STAT proteins (Fig. 8, compare lanes 2, 3 with lane 1). Because BMP4 causes astrocytic differentiation
in EGF-expanded stem cells, activation of STATs by combinations of
these factors was determined. There was no enhancement of STAT
activation caused by BMP4 or CNTF in the presence of EGF (Fig. 8).
These data suggest that the astrocytic differentiation effects of EGF
and BMP4 are not mediated through JAK-STAT activation.
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Figure 8.
EGF and BMP4 do not activate STAT proteins. Stem
cell cultures were treated with the mentioned factors for 15 min, and
nuclear extracts were prepared. EMSA assays were performed to determine
the extent of activation of STAT proteins as described in Materials and
Methods and the legend to Figure 2. STAT complexes are marked
a and b. EGF and BMP4 do not activate
STAT proteins.
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DISCUSSION |
FGF-expanded stem cells maintained in monolayers are multipotent
and give rise to astrocytes, oligodendrocytes, and neurons on
withdrawal of the mitogen (McKay, 1997). All the experiments described
here were performed on cultures that were in first or second passage.
The cells in these cultures are homogeneous by morphological criteria
and nestin immunohistochemistry. It is thus an excellent primary cell
culture system in which to study the biochemical mechanisms of
differentiation of CNS cells and, particularly, for defining the
biochemical basis for the transition from a multipotent stem cell to a
committed glial progenitor.
CNTF treatment leads to a prolonged activation of the JAK-STAT pathway
and transient activation of the MAPK pathway. The requirement of these
pathways in astrocytic differentiation was shown by blocking activation. A dominant negative Stat3 that prevents DNA binding of
activated Stat3 homodimers and heterodimers completely blocks astrocytic differentiation, whereas blocking activation of MAPKK, the
upstream activator of MAPK, causes a delay in astrocytic
differentiation by CNTF. Overexpression of MAPKK causes the converse
effect, a transient appearance of GFAP. Thus, the MAPK pathway
functions in the initiation of astrocytic differentiation, whereas
activation of Stat3 is required for complete differentiation. Stat3
activation as seen by EMSA occurs within 15 min of CNTF treatment. It
is likely to be transcriptionally active at this time, thus indicating a function for this protein early in the differentiation process.
Our data suggest that there is interaction between the MAPK and
JAK-STAT pathways during CNTF-mediated differentiation. The MAPK
pathway has been shown to have an effect on STAT activation (Zhang et
al., 1995; Chung et al., 1997) and on transactivation by STATs (Wen et
al., 1995). The experiments shown here indicate that the MAPK pathway
is not involved in CNTF-mediated STAT activation in FGF-expanded stem
cells. Perhaps interaction between the two pathways occurs at the level
of the GFAP promoter through an MAPK-sensitive transcriptional
regulatory site. In the MAPKK overexpression experiment it appears that
activation of MAPK causes a measure of astrocytic differentiation. FGF
also causes MAPK activation but does not cause astrocytic
differentiation. This may be a consequence of different levels of MAPK
activation in FGF-treated and virus-infected cells. However, it may
also be the case that FGF stimulates other pathways that inhibit
astrocytic differentiation. The different response of the cells to FGF
and CNTF is probably the result of the activation of distinct pathways
in response to the two factors.
CNTF and LIF have been implicated in astrocytic differentiation in two
experimental models. CNTF causes O2A progenitor cells to form
astrocytes in vitro (Hughes et al., 1988, Lillien et al., 1990). The number of astrocytes was greatly reduced in E18.5 LIFR null animals when compared with a wild-type littermate, implicating LIF
in either the survival or differentiation of astrocytes (Ware et al.,
1995). In a related model, LIF may be involved in the appearance of
astrocytes after CNS injury, because LIF expression is upregulated
30-fold within 24 hr after injury to the CNS (Banner et al., 1997). It
is interesting to speculate that the mechanisms by which astrocytic
differentiation occurs during embryonic development may be reactivated
in an adult cell during injury.
Our results contrast with that of Bonni et al. (1997), who have
recently suggested that the JAK-STAT pathway alone is instrumental in
causing CNTF-mediated astrocytic differentiation. In a
transfection-luciferase assay, inhibition of MAPK activation enhances
activation from a transfected GFAP promoter. An explanation for this
discrepancy is that the cultures being used in the two studies are
different. The data presented here were obtained on a homogeneous
population of cells that consistently gave 50-60% astrocytic
differentiation after 2 d and ~98% after 4-6 d of CNTF
treatment. Because the cultures of Bonni et al. (1997) are 60%
neuronal, the results of any biochemical assay in which the entire
population of cells is used may reflect neuronal rather than stem cell
responses.
Since GFAP is robustly and specifically induced in astrocytes, it may
be used as a paradigm for transcriptional regulation. It remains to be
seen whether the STAT sites in the GFAP promoter are functional, and if
so, whether they are equivalent. Also, it remains to be determined
whether sites corresponding to transcription factors activated by
MAPK-related pathways such as AP1 and CREB, which are present in this
promoter, are functional (Besnard et al., 1991; Kahn et al., 1997). In
other systems interaction between the MAPK-related and STAT pathways
has been shown to occur through integrators such as the p300 and
CREB-binding proteins (Horvai et al., 1997). It will also be important
to determine whether SMAD proteins interact with the GFAP promoter
either directly or indirectly. The prolonged activation of STAT for
8 d in the stem cells is in contrast to the brief activation
usually seen in vitro (Symes et al., 1994; Rajan et al.,
1996) but is reminiscent of LIF-induced in vivo STAT
activation in injured peripheral neurons (Rajan et al., 1995). In the
experiment shown in Figure 2, cells were treated every 24 hr for time
points >24 hr. However, Stat1 and Stat3 activation was seen even 72 hr
after one dose of CNTF (data not shown).
EGF promotes astrocytic differentiation, but it is not identical in its
effects to CNTF. In a clonal assay done with FGF-expanded cells,
mitogen withdrawal causes ~10% of cells to differentiate into
astrocytes (Johe et al., 1996). A similar experiment done with
EGF-expanded cells causes the number of astrocytes to increase to 50%
(Johe et al., 1996). In another experiment performed along the same
lines, FGF-expanded cells were treated with EGF for the final passage,
and both mitogens were withdrawn. This scenario also yields 50%
astrocytes (K. Johe, T. Hazel, and R. D. G. McKay, unpublished results). Thus, exposure to EGF just before mitogen withdrawal causes the cells to have a greater propensity to
differentiate into astrocytes. However, it is not instructive in its
action like CNTF. Although EGF has been reported to activate STATs (Fu and Zhang, 1993; Schindler and Darnell, 1995), in the experiments reported here there is no evidence for STAT activation after EGF treatment. Although we have not investigated the possibility of STAT
activation after EGF withdrawal, it seems highly unlikely. We conclude
that EGF promotes astrocytic differentiation by mechanisms that do not
include activation of STATs. BMP4 treatment causes astrocytic
differentiation in EGF-expanded precursors isolated from the murine E17
subventricular zone grown in neurosphere cultures (Gross et al., 1996).
This effect is not seen in our cells, which are isolated from E14
cortex and maintained in monolayers. Instead they respond to BMP4 by
differentiating into neural crest progeny (Hazel et al., 1997). BMP4
also does not cause activation of STAT proteins in our system.
Thus, both EGF and BMP4 cause astrocytic differentiation in different
paradigms. Both of these factors signal through SMAD proteins. BMP4
causes direct activation of the SMAD1 protein by receptor-mediated
phosphorylation (Lagna et al., 1996; Liu et al., 1996), whereas EGF
causes inhibition of transcriptional activation by the SMADs via the
MAPK pathway (Kretzschmar et al., 1997). Astrocytic differentiation in
both the EGF withdrawal and BMP4 treatment paradigms may be regulated
positively by the SMAD proteins. In the case of BMP4 this would occur
through direct activation, whereas withdrawal of EGF may cause an
alleviation of SMAD inhibition, leading to astrocytic differentiation.
These results are consistent with a model in which the involvement of
either STAT or SMAD signaling is necessary for astrocytic
differentiation depending on the type of stem cell undergoing
differentiation and the ligand causing it. In the experimental models
discussed here, CNTF causes astrocytic differentiation of FGF-expanded
CNS stem cells by STAT activation, whereas EGF and BMP may cause
astrocytic differentiation in their respective models via the SMAD
pathway. It is possible that FGF primes the cells for the
differentiation effects of CNTF in the model studied in this report,
whereas EGF exposure is required in the model of Gross et al. (1996)
for astrocytic differentiation in response to BMPs. Thus different
growth factors may activate distinct groups of transcription factors,
providing a method for plasticity in the timing, quantity, and quality
of astrocytes generated in the adult brain.
| |
FOOTNOTES |
Received Nov. 17, 1997; revised Feb. 19, 1998; accepted Feb. 20, 1998.
We thank Drs. M. Molne, L. van Grunsven, and M. Brenner for helpful
discussions, E. Saphier for technical assistance, Dr. J. E. Darnell Jr (Rockefeller University) for the Stat3 plasmids, and Dr. Y. Gotoh (Kyoto University, Kyoto, Japan) for the MAPKK virus vector.
Correspondence should be addressed to Ronald D. G. McKay,
Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room
5A29, Bethesda, MD 20892-4157.
| |
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Top
Abstract
Introduction
