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The Journal of Neuroscience, June 1, 2000, 20(11):4081-4090
Ciliary Neurotrophic Factor and Stress Stimuli Activate the
Jak-STAT Pathway in Retinal Neurons and Glia
Ward M.
Peterson,
Quan
Wang,
Roumiana
Tzekova, and
Stanley J.
Wiegand
Regeneron Pharmaceuticals, Tarrytown, New York 10591
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ABSTRACT |
Ciliary neurotrophic factor (CNTF) is pleiotrophic for central,
peripheral, and sensory neurons. In the mature retina, CNTF treatment
enhances survival of retinal ganglion and photoreceptor cells exposed
to otherwise lethal perturbation. To understand its mechanism of action
in vivo, the adult rat retina was used as a model to
investigate CNTF-mediated activation of Janus kinase/signal transducer
and activator of transcription (Jak-STAT) and ras-mitogen activated
protein kinase (ras-MAPK). Intravitreal injection of Axokine, an analog
of CNTF, phosphorylates STAT3 and MAPK and produces delayed
upregulation of total STAT3 and STAT1 protein in rat retina. Activated
STAT3 is predominantly localized in nuclei of retinal Müller
(glial) cells, ganglion cells, and astrocytes, but not in
photoreceptors. Although CNTF  -receptor (CNTFR) mRNA and protein
are localized predominantly if not exclusively in retinal neurons,
coincident CNTF-mediated STAT3 signaling was observed in both glia and
neurons. CNTF-induced activation of Jak-STAT signaling prompted us to
investigate STAT3 phosphorylation after a variety of stress-mediated,
conditioning stimuli. We show that STAT3 is activated in the retina
after exposure to subtoxic bright light, mechanical trauma, and
systemic administration of the 2-adrenergic agonist
xylazine, all of which have been shown previously to condition
photoreceptors to resist light-induced degeneration. These results
demonstrate that CNTF directly stimulates Jak-STAT and ras-MAPK
cascades in vivo and strongly suggest that STAT3
signaling is an underlying component of neural responsiveness to stress
stimuli. The observation that CNTF activates STAT3 in ganglion cells,
but not in photoreceptors, suggests that Jak-STAT signaling influences
neuronal survival via both direct and indirect modes of action.
Key words:
ciliary neurotrophic factor; signal transducer and
activator of transcription; Müller cell; ganglion cell; glial
fibrillary acidic protein (GFAP); mitogen-activated protein kinase; extracellular receptor kinase; neuroprotection; conditioning
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INTRODUCTION |
The mature nervous system responds
to sublethal environmental stress and injury by mounting a
self-protective mechanism that enhances neuronal survival in the face
of subsequent insult. For example, retinal photoreceptors in albino
rats can be conditioned to resist the damaging effects of prolonged
exposure to constant light if animals are previously exposed to a
conditioning period of nondamaging, bright light (Liu et al., 1998 ).
This phenomenon of conditioning in photoreceptors and other neurons is
thought to involve stress-mediated release of growth factors and
cytokines and subsequent activation of intracellular signaling pathways (Mattson and Scheff, 1994). Ciliary neurotrophic factor (CNTF) is one
such neurocytokine with pleiotrophic actions in the developing and
mature nervous system (Adler et al., 1979; Sendtner et al., 1994; Segal
and Greenberg, 1996). CNTF is a cytosolic protein that lacks a
consensus sequence for secretion and has been postulated to act as an
injury-activated factor under pathological conditions (Lin et al.,
1989; Stockli et al., 1989). Normally localized inside Schwann cells,
CNTF can be detected extracellularly after sciatic lesion (Sendtner et
al., 1992). In mature retina, CNTF is upregulated in vivo in
response to a variety of stress-mediated conditions (Cao et al., 1997;
Liu et al., 1998; Ju et al., 1999). Exogenous CNTF protects mature
neurons in the CNS and peripheral nervous system from
degeneration arising from multiple etiologies (LaVail et al., 1992;
Sendtner et al., 1992; Hagg and Varon, 1993; Anderson et al.,
1996).
CNTF binds to its -receptor (CNTFR) and two signal-transducing
transmembrane subunits, LIFR and gp130, thus specifically activating the Janus tyrosine kinase/signal transducers and activators of transcription (Jak-STAT) signaling pathway (Stahl et al., 1994; Stahl and Yancopoulos, 1994). The activated CNTF receptor complex preferentially phosphorylates STAT3 at residue Y705 and to a lesser extent STAT1 at Y701, thus promoting homodimerization or
heterodimerization of STAT3 and STAT1 and subsequent nuclear
translocation of dimerized STAT proteins (Bonni et al., 1993; Wegenka
et al., 1993; Darnell et al., 1994). CNTFR is tethered to the plasma
membrane by glycosylphosphatidylinositol linkage and can be
released from the cell, particularly under pathological conditions.
Soluble CNTFR can bind CNTF and signal via the LIFR/gp130
heterodimer in vitro (Davis et al., 1993); CNTFR may
therefore, under some conditions, function as a soluble mediator of the
effects of CNTF in vivo. Although CNTFR mRNA is expressed
predominantly, if not exclusively, by neurons in the normal developing
and mature brain and retina (Kirsch and Hofmann, 1994; MacLennan et
al., 1996; Kirsch et al., 1997; Fuhrmann et al., 1998), cellular
localization of CNTF-mediated activation of the Jak-STAT pathway has
not been demonstrated in vivo. In the present study, the
adult rat retina was used as a model system to investigate CNTF- and
stress-mediated Jak-STAT signaling in neurons and glia.
The involvement of the ras-mitogen activated protein kinase (MAPK)
pathway in response to CNTF administration was also investigated at the
cellular level in the present study and compared with Jak-STAT signaling. We show that STAT3 is activated in both retinal neurons and
glia, whereas p42/44 MAPK is activated only in retinal glia. Additionally, we demonstrate that a variety of stress-mediated, conditioning stimuli also activate Jak-STAT signaling in mature retina.
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MATERIALS AND METHODS |
Protein and antibodies. The potency of human CNTF is
increased by substituting glutamine at position 63 with an arginine
(Panayotatos et al., 1993). Additionally, C-terminal truncations of up
to 27 amino acids of CNTF were found to be at least as active as
full-length CNTF (Negro et al., 1994). Axokine is a modified version of
human CNTF in which the C terminus has been truncated by 15 residues, the free cysteine has been replaced with an alanine, and the glutamine at position 63 has been switched to an arginine. Axokine is three to
five times more potent than the CNTF parent molecule as measured by
in vitro neuronal survival assays and in vivo
studies and has improved stability properties over CNTF. Polyclonal
antibodies against CNTF were raised by immunizing rabbits with whole
Axokine protein. Antibodies from bleeds were affinity purified and used in immunoblots at a dilution of ~1 µg/ml and were shown to
cross-react with endogenous rat CNTF in Western blots and
immunocytochemistry. Immunoblots were performed with commercially
available antibodies for STAT3 and Tyr705-phosphorylated STAT3
(pSTAT3), STAT1 and Tyr701-phosphorylated STAT1 (pSTAT1), and p44/42
MAPK and Thr202/Thr204 phospho-p44/42 MAPK (pMAPK) (New England
Biolabs, Beverly, MA). Immunocytochemical studies were performed with
the same STAT3 and pSTAT3 antibodies. Additional immunocytochemical
studies used a monoclonal Cy3-conjugated anti-glial fibrillary acidic
protein (GFAP) and a monoclonal anti-S-100 (-subunit) (Sigma,
St. Louis, MO).
Animal handling and surgery. Male Sprague Dawley rats,
10-14 weeks of age, were used in all experiments. Animals were
entrained in a 12 hr light/dark cycle with an in-cage illuminance of
<25 foot-candles for at least 5 d before experiments.
Unless otherwise specified, animals were anesthetized with an
intraperitoneal injection of a mixture of chloral hydrate and
pentobarbital (CPB) (260 mg/kg chloral hydrate and 50 mg/kg sodium
pentobarbital by body weight). Axokine or vehicle (10 mM phosphate buffer) was injected (1 µl) into
the vitreal cavity of the rat eye using a Hamilton (Reno, NV)
microliter syringe attached to a beveled 32 gauge Hamilton needle. The
injection was made by inserting the needle through the sclera ~0.3 mm
posterior to the limbus near the superior pole of the eye and guiding
the needle ~1.5 mm into the vitreal cavity at an angle that avoids
the lens. This injection procedure also avoided penetration of and
trauma to the neural retina. Animals were killed by either intracardial
overdose of anesthesia or exanguination of deeply anesthetized animals,
transcardial perfusion of heparinized 0.9% NaCl Ringer's solution,
followed immediately by rapid, ice-cold perfusion in PBS
containing 4% paraformaldehyde. All experiments on animals were
conducted in accordance with the Policy on the Use of Animals in
Neuroscience Research.
Conditioning protocols. Retinas were assayed for STAT3
activation in response to three separate stress-mediated, conditioning protocols that have been shown previously to protect photoreceptors from the damaging effect of prolonged bright light. The first conditioning stimulus involved the exposure of animals to a period of
subtoxic bright light that does not damage the retina but activates an
endogenous neuroprotective mechanism in photoreceptors (Liu et al.,
1998). In brief, rats were brought in-house, entrained for 1-2 weeks,
and then exposed to 125-150 foot-candles for either 24 or 48 hr before
returned to normal cyclic lighting conditions for up to 1 week. Retinas
were collected immediately and at 1, 3, and 7 d after exposure to
the conditioning period of bright light. In a second conditioning
paradigm, the retina was mechanically injured by inserting a 30 gauge
needle through the sclera and directly into the retina at ~1-2 mm
posterior to the pars plana in the superior hemisphere. After
mechanical injury, animals were then returned to normal cyclic lighting
conditions for 7 d before being killed. In a third
conditioning protocol, each animal was given a single intraperitoneal
injection of ketamine alone (42 mg/kg), xylazine alone (8.4 mg/kg), or
ketamine-xylazine combination (42 mg/kg ketamine and 8.4 mg/kg
xylazine). Previous work has shown that daily intramuscular injections
of the 2-adrenergic agonist xylazine for
4 d significantly attenuates photoreceptor damage caused by
subsequent exposure to 7 d of constant light (Wen et al.,
1996).
Immunoblot analysis. Fresh retinas were harvested
immediately after the animals were killed, snap frozen in liquid
nitrogen, and stored at  80°C. Retinas were homogenized in
lysis-phosphatase inhibitor buffer containing: 20 mM Tris-HCl, 2 mM EGTA, 150 mM NaCl, 2 mM
Na3VO4, 1 µM okadaic acid, 50 mM
NaF, 1% NP-40, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml
pepstatin-A, and 1 mM PMSF. Protein levels were
quantified using BCA protein assay (Pierce, Rutherford, IL). Total
protein of 50 µg from each sample was electrophoresed (Mini-Protean
II system; Bio-Rad, Hercules, CA) on polyacrylamide gels and
transferred to nitrocellulose membranes (Stratagene, La Jolla, CA). For
STAT3/pSTAT3, STAT1/pSTAT1, and MAPK/pMAPK paired immunoblots, protein
samples were loaded in duplicate on separate gels and immunoblotted
under identical conditions. All of the primary antibodies were applied
at 1:1000 dilution. Signals were visualized using a Photope-HRP
Detection Kit for Western Blotting Signals (New England Biolabs).
Signal was quantified by conventional densitometric analysis (NIH Image software).
Immunohistological analysis. Retinal sections were stained
for STAT3, pSTAT3, MAPK, pMAPK, GFAP, and S-100 (-subunit) antigen. Eyes were cryoprotected with 30% sucrose, frozen and embedded in
Tissue-TEK OCT compound (Miles, Elkhart, IN), cryosectioned (10 µm),
and mounted onto Probe-on Plus glass slides (Fisher Scientific, Pittsburgh, PA). For the mechanically injured eyes, care was taken to
ensure that retinal sections included the region surrounding the site
of focal injury. Retinal sections were stained for STAT3, pSTAT3, MAPK,
pMAPK, GFAP, and S-100 antigen, all at a dilution of 1:200.
Biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA)
was used as secondary antibody (diluted 1:1000), and staining was
completed using an avidin-biotin-peroxidase (ABP) complex reaction
(Vectastain Elite ABC kit; Vector Laboratories). The peroxidase was
visualized with diaminobenzidine as the chromagen. For
double-labeling experiments using S-100 antigen and pSTAT3, sections
were first processed for pSTAT3 and visualized using the ABP method,
and then reblocked and stained for S-100 antigen. S-100 labeling was
visualized with a Cy3-conjugated sheep anti-mouse IgG antibody (diluted
1:200; Sigma).
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RESULTS |
Axokine-induced phosphorylation of STAT3 and STAT1 in total
rat retina
Antibodies that recognize total STAT3 and Y705-phosphorylated
STAT3 (pSTAT3) were used for immunoblotting homogenized rat retina.
Expression of total STAT3 or pSTAT3 was evaluated at time points
ranging from 5 min to 7 d after a single intravitreal injection of
Axokine (1 µg) or vehicle (10 mM phosphate buffer), or
after administration of anesthesia (CPB) alone (Fig.
1A). STAT3 is normally inactive but can be phosphorylated as early as 15 min after an injection of Axokine. The levels of phosphorylated STAT3 increased over
the following 2 d and disappeared by 4 d. Intravitreal
injection of vehicle resulted in only slight phosphorylation of STAT3,
beginning 1-4 hr after the injection and persisting for 16 hr. A
similar pattern of pSTAT3 activation was seen in the anesthesia control group. An increase in total STAT3 protein was observed only in the
Axokine-treated group. This increase in total STAT3 is evident by 16 hr, most pronounced at 2 d, and is maintained for at least 1 week
(Fig. 1C). This secondary increase in total and
unphosphorylated STAT3 levels may be mediated by the earlier robust
phosphorylation of STAT3.
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Figure 1.
Effects of Axokine on phosphorylated and total
STAT3, STAT1, and MAPK in homogenized rat retinas. A,
Retinas were harvested at various times indicated (5 min to 7 d)
after a single intravitreal injection of Axokine. Homogenized
retinas were Western blotted (50 µg/lane) to detect
total STAT3 and Y705-phosphorylated STAT3 (pSTAT3). The band size for
both STAT3 and pSTAT3 is the expected 92 kDa. Axokine produced robust
pSTAT3 signal beginning at 15 min and disappearing by 4 d. Weaker
and more transient pSTAT3 signals were seen in the vehicle and
anesthesia (CPB) control groups. B, Membranes from
Axokine-treated STAT3/pSTAT3 immunoblots were stripped and reblotted
using an antibody that recognizes both CNTF and Axokine (23 and 21 kDa,
respectively). Axokine was detected in the retina for up to 2 d
after injection. C, Graph summarizing the time-dependent
effects of total STAT3 for each treatment group. STAT3 signal was
quantified at each time point using conventional densitometric analysis
and normalized to the STAT3 signal at 5 min. Analysis of immunoblots
from time course studies culled from three separate groups of animals
shows a significant increase in normalized STAT3 signal in the
Axokine-treated group (p < 0.05; repeated
ANOVA, Dunnett's post hoc t
test). Error bars are in terms of SE. D,
Immunoblots for total STAT1 and Y701-STAT1 (pSTAT1) were performed on
Axokine-treated and anesthesia control retinas. Axokine weakly
activated pSTAT1 at the later time points but dramatically increased
total STAT1 levels by 16 hr. E, Western blots for total
MAPK and pMAPK for Axokine-treated retinas showed robust transient
activation of MAPK at 60 min.
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Membranes that were immunoblotted with either STAT3 or pSTAT3
antibodies were stripped and reprobed for CNTF and Axokine using a
polyclonal antibody that recognizes both proteins (Fig.
1B). Axokine could be detected in retinal samples
between the 5 min and 2 d time points, which is coincident with
the appearance of the pSTAT3 bands in the Axokine-treated retinas in
Figure 1A. No apparent changes in the levels of
endogenous CNTF were observed in any of the three treatment groups.
Total STAT1 and Y701-phosphorylated STAT1 (pSTAT1) proteins were also
immunoblotted by using antibodies that discriminated between these
forms of STAT1. Although STAT1 is normally present in retinal
homogenates, Axokine injection did not result in immediate phosphorylation of STAT1 (Fig. 1D). However, a small
of amount of pSTAT1 was detected at 16 hr and 2 d after Axokine
treatment, with lower levels also 16 hr after anesthesia alone.
Although we saw no phosphorylation of STAT1 at the earlier time points, Axokine nevertheless produced a delayed, but marked, increase in total
STAT1. As for total STAT3, an increase in total STAT1 protein was noted
by 16 hr, peaked at 2 d, and persisted through day 7 in the
Axokine group.
The ras-MAPK pathway represents a distinct intracellular signaling
cascade that has been shown previously to be activated in the nervous
system by a variety of neurotrophic factors, including the nerve growth
factor family of neurotrophins, and by a variety of
stress-mediated stimuli (Segal and Greenberg, 1996). To determine whether CNTF activates the ras-MAPK pathway, we used antibodies that
discriminate between total and phosphorylated p44/42 MAPK (pMAPK) to
detect activated p44/42 MAPK after Axokine injection. Immunoblotting
results for Axokine- and vehicle-treated control retinal homogenates
are shown in Figure 1E. Axokine treatment produced a
much more robust phosphorylation of MAPK at 60 min than vehicle
treatment, and by 4 hr, pMAPK signal was significantly reduced.
Our results indicate that a single intravitreal injection of Axokine
activates two principal tyrosine kinase signaling pathways in the
retina, Jak-STAT and ras-MAPK. Axokine produced robust STAT3
phosphorylation within 15 min of injection, which persisted for at
least 2 d. Axokine did not activate STAT1 until the 16 hr time
point. Delayed upregulation of total STAT1 and STAT3 also was observed
after Axokine injection but not under control conditions. Termination
of both STAT1 and STAT3 phosphorylation signals after 2 d is
coincident with clearance of Axokine from the retina. In contrast,
Axokine-dependent phosphorylation of MAPK was observed only at the 1 hr
time point, and no change in the levels of total MAPK was observed
throughout the duration of the study.
Immunolocalization of basal and activated total STAT3
At 15 min after vehicle injection, light diffuse staining of total
STAT3 was observed in the ganglion cell layer (GCL), in radial
processes within the inner plexiform layer (IPL), in a few cell bodies
of the inner nuclear layer (INL), and laterally throughout the outer
plexiform layer (OPL) (Fig.
2A). Similar patterns
of STAT3 immunoreactivity were observed in untreated eyes (results not
shown).
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Figure 2.
Immunolocalization of total STAT3 in vehicle- and
Axokine-treated retinal cross-sections viewed with differential
interference contrast optics. A, Injection of vehicle
(VEH) buffer 15 min before the animals
were killed exhibited the same STAT3 labeling pattern as untreated eyes
(latter not shown). In the inner retina, diffuse labeling of STAT3 was
seen along the NFL and GCL, and along radial processes within the IPL
and INL (thin arrow). Weak STAT3 immunoreactivity was
apparent in the OPL but not in the photoreceptor layer.
B, Within 15 min of Axokine (AXK)
injection, we observed intense labeling of nuclei along the NFL
(thick arrow), heavier labeling of GC bodies and nuclei
(arrowhead), and heavier labeling of cell bodies and
nuclei in the INL (thin arrow) near the injection site.
C, No specific labeling was observed in the absence of
primary antibody. D, Retinal cross-section taken
from four contiguous regions, extending from near the injection site
(right half of panel) to further
away (left half of panel).
E-H, Magnification of boxed regions of
D showing spatial profile of STAT3 labeling from regions
distal to the injection site (E) to regions
closest to the injection site (H).
Proximity to the injection site is associated with a progressive
increase in STAT3 labeling of cell bodies and nuclei in INL and a
concomitant gradual decrease of labeling in processes in the IPL,
suggesting mobilization of STAT3 from cytoplasm to cell
body, and then to nucleus. Heavy labeling of nuclei in the NFL and GCL
is seen throughout the entire region. RPE, Retinal
pigment epithelium; OS, photoreceptor outer
segments; IS, photoreceptor inner segments.
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As early as 15 min after Axokine injection, a change in the
distribution of total STAT3 could be observed in a limited region of
the retina: (1) appearance of dense staining of nuclear profiles along
the inner limiting membrane (ILM) and nerve fiber layer (NFL) in which
retinal astrocytes are localized; (2) increased labeling of retinal
ganglion cell bodies; and (3) appearance of dense labeling of scattered
cell bodies and nuclei within the INL (Fig. 2B). In
other cryosections from the same eye, a coincident increase in Axokine
labeling was observed in this same limited region of the retina
(results not shown). In the rest of the retina, total STAT3
immunoreactivity was similar to that of untreated eyes. Our results
suggest that the changes in STAT3 labeling occur first in regions
proximal to the injection site.
After 30 min of Axokine treatment, a characteristic gradient of total
STAT3 labeling was observed within the retina (Fig. 2D). In regions away from the injection site (Fig.
2D, far left, E), we observed
dense labeling of nuclei in the nerve fiber layer and some labeling of
ganglion cell bodies, with STAT3 labeling in the other retinal layers
remaining essentially the same as that found in untreated eyes.
However, with increasing proximity to the injection site, we observed a
gradual disappearance of labeling of processes in the IPL and a
concomitant increase in nuclear-specific immunoreactivity in the INL
(Fig. 2D, middle and far right,
F-H). At 60 min after Axokine injection, a
consistent labeling pattern for STAT3 was observed throughout the
entire retina that is similar to the 30 min STAT3 labeling near the
injection site (results not shown). The STAT3 labeling in the 30 and 60 min vehicle-treated eyes is similar to that of untreated eyes (results
not shown). We saw no evidence for STAT3 labeling in photoreceptors
under any of our experimental conditions.
Immunolocalization of phosphorylated STAT3
As early as 15 min after Axokine injection, heavy labeling of
nuclei in the NFL and GCL was observed (Fig.
3B). Vehicle-treated retinas
exhibited no specific pSTAT3 staining at this time point (Fig.
3A). pSTAT3 labeling was strongest at the 30 and 60 min time
points after Axokine injection and was localized primarily to nuclei in
the NFL, GCL, and INL (Fig. 3C-E). Thus, the temporal and
spatial labeling profile for pSTAT3 very closely matches that of total
STAT3.
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Figure 3.
Effects of Axokine on pSTAT3 immunolocalization
and colocalization of pSTAT3 and S-100 in Müller cells.
A, In 15 min vehicle-treated (VEH)
eyes, sparse pSTAT3 labeling is present only along the NFL.
B, Labeling of astrocytes and GC nuclei is observed
adjacent to the injection site 15 min after Axokine
(AXK) treatment. C,
D, Two representative regions of pSTAT3 immunoreactivity
in the same retina 30 min after Axokine treatment. C,
pSTAT3 heavily labeled GC nuclei and weakly labeled nuclei in the INL.
D, pSTAT3 heavily labeled nuclei in the INL but produced
little labeling in GC bodies and nuclei. E, At 1 hr,
strong labeling of nuclei in INL (thin arrow), GCs
(thick arrow), and astrocytes (arrowhead)
was observed in most of the retina. F, A diffuse band of
pSTAT3 labeling was evident along the GCL and NFL in vehicle-treated
eyes. G, pSTAT3 labeling of nuclei in the
INL gradually disappeared by 4 hr after Axokine treatment, but signal
could still be detected in nuclei within the GCL. H, At
16 hr after Axokine injection, no specific pSTAT3 immunoreactivity was
detected. I, J, One-to-one colocalization
of S-100 antigen (left) and pSTAT3
(right) immunoreactivity in the INL is consistent with
exclusive localization of STAT3 in Müller cells in the INL.
K, At 60 min after Axokine treatment, pSTAT3
immunoreactivity was observed in nuclei of astrocytes throughout the
optic nerve head (ONH). RPE,
Retinal pigment epithelium; OS, photoreceptor outer
segments; IS, photoreceptor inner segments.
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pSTAT3 immunoreactivity was observed in the GCL in 4 hr Axokine-treated
and vehicle-treated groups (Fig.
3F,G). However, we saw no pSTAT3
labeling in the 16 hr Axokine group (Fig. 3H),
despite robust pSTAT3 signal in the immunoblots at 16 hr after Axokine injection. The absence of pSTAT3 labeling at these later time points
was not attributable to an artifact in the processing, because
positive pSTAT3 controls (i.e., 30 min or 1 hr Axokine-treated eyes)
were run in tandem from the same experimental group of animals, processed with identical immunohistological procedures, and yielded robust pSTAT3 immunoreactivity. These results suggest that, at these
later time points, the antigen recognition site on pSTAT3 is not
accessible for binding to the antibody in whole, freshly fixed tissues
but is accessible under the denaturing conditions of a Western blot
(see Discussion).
The results of total STAT3 and pSTAT3 immunostaining studies are
consistent with cytoplasmic localization of STAT3 under untreated conditions, followed by phosphorylation and nuclear translocation of
pSTAT3 in retinal astrocytes, ganglion cells, and Müller cells after Axokine treatment. To confirm the notion that pSTAT3 labeling in
the INL is localized exclusively in Müller cells, we show that
pSTAT3 is specifically colocalized with a Müller cell marker, S-100 (-subunit) antigen, in the INL (Fig.
3I,J). In addition, intense
nuclei-specific pSTAT3 immunoreactivity was observed in the nuclei of
astrocytes distributed along the NFL and optic nerve head (Fig.
3K) at 1 hr after Axokine injection but not in
vehicle-treated eyes.
Immunolocalization of phosphorylated MAPK
The immunoblots for pMAPK from Figure 1E
demonstrated that Axokine treatment also activated p42/44 MAPK. The
same pMAPK antibodies were used to determine cell-specific localization
of pMAPK after Axokine or vehicle treatment (Fig.
4). At 30 min after Axokine injection,
the regions proximal to the injection site showed intense pMAPK
labeling in cells and processes along the ILM, in cells within INL, and
in radial processes within the IPL and the outer nuclear layer (ONL)
(Fig. 4A). Strong pMAPK staining was also observed
surrounding the inner retinal vasculature. This labeling pattern is
consistent with distribution of pMAPK in astrocyte and Müller
cell processes, cell bodies, and nuclei. By 60 min, pMAPK labeling in
Müller cell processes permeated the ONL and distributed along the
microvillous extensions along the outer limiting membrane (Fig.
4C). Specific phospho-MAPK labeling in vehicle-treated eyes
was very weak and primarily confined to the vicinity of the
injection site (Fig. 4B,D). No
photoreceptor-specific labeling of pMAPK was observed in either
Axokine- or vehicle-treated eyes.
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Figure 4.
Immunolocalization of pMAPK. A, At
30 min after Axokine (AXK) injection, intense
pMAPK staining is noted in cells and cell processes in the NFL,
particularly surrounding blood vessels, which is consistent with
staining of astrocytes. Intense staining of nuclei in the INL and of
radial elements extending from the NFL through the ONL is consistent
with distribution of pMAPK throughout Müller cell bodies and
processes. At this time point, pMAPK staining was strongest in regions
nearest the injection site. B, Significantly weaker
pMAPK staining along the NFL in vehicle-treated
(VEH) eyes in regions proximal to the injection
site suggests stress-induced activation of MAPK in astrocytes. Very
little staining of pMAPK was observed in Müller cells.
C, Representative photomicrograph of the uniform pMAPK
staining apparent within the entire retina at 60 min after Axokine
injection. Note the appearance of pMAPK labeling along the outer
limiting membrane. D, Very little labeling of pMAPK was
observed in 1 hr vehicle-treated eyes.
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Time course of Axokine-induced GFAP expression in
Müller cells
To compare the time course of Axokine-induced expression of
GFAP in Müller cells with that of STAT3 activation, we injected Axokine into the vitreous and processed retinas for GFAP
immunohistochemistry 1 hr to 3 weeks later. GFAP is normally localized
in astrocytes along the ILM and NFL, and this labeling pattern was
unchanged for up to 16 hr after a single vehicle or Axokine injection
(Fig. 5). However, after 2 d, the
Axokine-treated eyes showed increased labeling of GFAP in Müller
cell processes along the IPL. This pattern of GFAP immunoreactivity was
most marked at 7 d after Axokine injection, whereas the
contralateral vehicle-treated eyes showed no upregulation or altered
distribution of GFAP (Fig. 5). The upregulation of GFAP in Müller
cells after Axokine treatment remained strong for at least 3 weeks
after the single injection of Axokine, whereas GFAP distribution in
contralateral vehicle-treated eyes remained unchanged during this same
period (results not shown). These results showing CNTF-mediated
phosphorylation of STAT3 in Müller cells therefore provide
evidence for a direct link between CNTF-mediated STAT3 activation and
GFAP expression in the same cell type in vivo.
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Figure 5.
Time course of Axokine-induced expression of GFAP
in Müller cells. At 16 hr, vehicle-injected
(VEH) and Axokine-injected
(AXK) retinas still showed essentially normal
GFAP labeling, restricted to astrocyte processes and end feet of
Müller cells along the NFL and surrounding blood vessels. At
2 d after Axokine injection, GFAP labeled radial processes in the
IPL. At 7 d, labeling of processes throughout the IPL and into the
ONL was observed in Axokine-treated eyes but not vehicle-treated eyes.
Labeling of radial processes throughout the IPL persisted for at least
3 weeks after Axokine treatment.
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Effects of stress-mediated stimuli on STAT3
Exposure to subtoxic bright light for a period of 12-48 hr,
followed by 3 d of normal cyclic lighting, elicits an endogenous neuroprotective response in Sprague Dawley rats that protects photoreceptors from damage upon subsequent exposure to toxic levels of
bright light (Liu et al., 1998). We have independently confirmed that
this conditioning exposure to bright light does not lead to
photoreceptor degeneration (results not shown). In animals that
received 24 hr of subtoxic levels of continuous illumination, phosphorylation of STAT3 was observed immediately after termination of
the light exposure and persisted for at least 4 d (Fig.
6A, top two
panels). Likewise, robust pSTAT3 was detected for 7 d after
48 hr of exposure to subtoxic bright light. In both conditioning paradigms, a notable increase in total STAT3 was observed immediately after continuous bright light exposure and persisted several days thereafter. In contrast, lower levels of pSTAT1 were detected after 24 and 48 hr of subtoxic bright light exposure (Fig. 6A, bottom two panels). Nevertheless, a striking increase in
total STAT1 was observed in both conditioning paradigms. These results indicate that STAT3 phosphorylation and upregulation of total STAT3 and
STAT1 levels occur and persist after a period of conditioning exposure
to subtoxic levels of bright light.
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Figure 6.
Activation of STAT3 and/or STAT1 after
conditioning with 24 or 48 hr of bright light or treatment with various
combinations of ketamine and xylazine. In A, animals
were recovered in normal lighting conditions for the time periods shown
before retinas were harvested for immunoblotting. A,
STAT3 was activated immediately after 24 or 48 hr of constant bright
light (day 0). An increase in total STAT3 levels was also observed in
both conditioning groups. Twenty-four and 48 hr of bright light
produced a small, delayed increase in pSTAT1 signal during the recovery
period and a robust increase in total STAT1 levels. B,
Effects of various combinations of ketamine and xylazine
(intraperitoneal injection) on pSTAT3 and total STAT3 signal in retina.
Xylazine alone or in combination with ketamine produced a robust
phosphorylation of STAT3 4 hr after administration. No activation of
STAT3 was observed in the ketamine group.
|
|
Repeated administration of agonists for
2-adrenergic receptors, such as xylazine and
clonidine, can also protect photoreceptors from light damage (Wen et
al., 1996). We observed that anesthetizing rats with ketamine and
xylazine phosphorylated STAT3 at 4 hr after the single injection of
anesthesia, but the pSTAT3 signal gradually disappeared by 2 d
(Fig. 6B). A similar effect on pSTAT3 was also seen
in animals that received xylazine alone, but not in animals that
received ketamine alone. We conclude that xylazine alone produces a
relatively robust, but short-lived, activation of STAT3.
Mechanical injury to the retina confers protection of photoreceptors
surrounding the site of injury from the damaging effects of prolonged
exposure to bright light. It has been shown that the retina undergoes a
variety of subcellular and biochemical changes in response to trauma
and injury, including upregulation of basic FGF, CNTF, GFAP, and
c-fos (Wen et al., 1995; Yoshida et al., 1995). We induced
mechanical trauma by producing a small injury in the superior retina
~2 mm from the limbus by inserting a 30 gauge needle through the
sclera. We then sectioned the eye 1 week later to examine GFAP, pSTAT3,
and total STAT3 labeling near and far from the site of trauma.
Increased GFAP and total STAT3 labeling was observed at and near the
site of injury and surrounded the region of trauma, but GFAP and STAT3
labeling remained relatively normal at regions of the retina distal
from the site of injury (Fig. 7). pSTAT3
labeling was not significantly above background levels (results not
shown). These results clearly demonstrate a dramatic increase in
cellular STAT3 expression near and surrounding the site of injury.
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Figure 7.
Needle injury to the retina induces GFAP
upregulation and increases total STAT3 labeling around the injury site.
A, Fluorescent photomicrograph of robust GFAP-positive
processes in the GCL (which borders the inner limiting membrane) and
throughout the retina encompassing the region of trauma.
B, GFAP labeling in the same eye in a region of the
retina that is far from the site of injury. Normal GFAP-positive
labeling in the region between the GCL and the ILM is observed.
C, In a representative section from the same region as
A, increased STAT3 signal is detected along ILM, the
GCL, and radial processes in the IPL, as well as cell bodies and nuclei
in the INL. D, In a representative section from the same
region as B, STAT3 immunoreactivity in regions far from
the injection site is similar to that of unperturbed retinas.
RPE, Retinal pigment epithelium; OS,
photoreceptor outer segments.
|
|
| |
DISCUSSION |
The present study demonstrates that the Jak-STAT signaling pathway
is activated in adult rat retina in response to an intravitreal injection of Axokine, an analog of CNTF. In addition, intraperitoneal delivery of the 2-agonist xylazine, exposure
to nondamaging bright light, and mechanical injury to the retina also
produce a similar response. All four of these stimuli have been shown
to protect retinal photoreceptors from the damaging effects of exposure
to prolonged periods of bright light (LaVail et al., 1992; Wen et al.,
1996; Liu et al., 1998). We also show that Axokine activates p44/42
MAPK, which confirms previous work demonstrating the involvement of
ras-MAPK signaling in response to neuronal stress and subsequent cell
survival (Segal and Greenberg, 1996; Fukunaga and Miyamoto, 1998).
Pronounced upregulation of endogenous, nonphosphorylated STAT3 and
STAT1 protein was observed at later time points after Axokine treatment
or after exposure (24-48 hr) to elevated levels of ambient light.
These results suggest a secondary phase of responsiveness, mediated by
both STAT3 and STAT1. The timing of this second phase coincides closely
with Axokine-induced upregulation of GFAP in Müller cells.
Immunocytochemical studies demonstrate that Axokine produces rapid,
persistent activation of STAT3 in retinal Müller cells,
astrocytes, and retinal ganglion cells. Although CNTF and these
stress-evoked stimuli all protect photoreceptors from environmental insult, we saw no evidence for activation of STAT3 or MAPK in the
photoreceptors themselves.
Localization of STAT3 and pSTAT3: relationship to CNTFR and
time course
In mature rat retina, CNTFR mRNA is found in horizontal cells
and subpopulations of amacrine and ganglion cells (Kirsch et al.,
1997). Here we show that, although Axokine activates STAT3 in retinal
ganglion cells, it also activates STAT3 in Müller (glial) cells
and astrocytes, which do not appear to express CNTFR. Conversely, we
saw no evidence for STAT3 activation in horizontal and amacrine cells.
A possible mechanism for CNTF-mediated signaling in retinal glial cells
may involve rapid, CNTF-mediated release of CNTFR from the plasma
membrane of horizontal, amacrine, and ganglion cells, and subsequent
activation of the CNTF tripartite receptor complex in Müller
cells and astrocytes. The localization of LIFR and gp130 in the
retina is currently not known. Our findings nevertheless demonstrate
that CNTF-mediated STAT3 signaling is not necessarily restricted to
cells that express appreciable levels of CNTFR and that cells that
express CNTFR mRNA do not invariably respond to CNTF. These
observations support the hypothesis that CNTFR may serve as soluble
mediator of the effects of CNTF in vivo (Davis et al.,
1993).
Within 30 min, Axokine administration resulted in phosphorylation of
STAT3 and a redistribution of STAT3 from the cytoplasm to the nucleus
in Müller cells, astrocytes, and ganglion cells. After 1 hr,
nuclei-specific pSTAT3 staining began to diminish. After 4 hr,
pSTAT3-positive cells were restricted to the ganglion cell layer in
Axokine-treated and vehicle-treated eyes. At 16 hr in Axokine-treated
eyes, pSTAT3-specific immunolabeling disappeared, although strong total
pSTAT3 signal was observed in immunoblots. The disparity between pSTAT3
immunoblot and immunostaining at the later time points raises the
possibility that the antibody recognition site for pSTAT3 is occluded
by associated protein(s) at some point after its translocation to the
nucleus. Recent work has shown that PIAS3, a member of the protein
inhibitor of activated STAT, specifically binds to
phosphorylated STAT3 and inactivates STAT3-mediated gene transcription
(Chung et al., 1997). Protein-protein interaction between PIAS3 and
the Y705-phosphorylated site of STAT3 may therefore account for the
absence of pSTAT3-specific labeling in aldehyde-fixed tissue sections.
Specificity of STAT3 verses STAT1 and relationship to MAPK
CNTF-mediated stimulation of STAT1 and STAT3 in neural precursor
and neuroepithelial stem cells promotes their differentiation into
astrocytes (Rajan et al., 1996; Bonni et al., 1997; Rajan and McKay,
1998). In stable, differentiated cells lines, such as COS or HeLa, CNTF
preferentially phosphorylates STAT3 rather than STAT1 (Boulton et al.,
1995; Stahl et al., 1995). The preferential activation of STAT3 by CNTF
has been attributed to alterations in modular tyrosine-based motifs in
cytokine receptors (Stahl et al., 1995). Our results indicate that
Axokine predominantly activates STAT3 rather than STAT1 in the mature
retina and support the notion that STAT3 is the primary effector
protein of CNTF-mediated Jak-STAT signaling. A secondary signaling
event, perhaps mediated by STAT3 feedback to STAT1, may be responsible
for the delayed and weaker CNTF-mediated phosphorylation of STAT1 and
subsequent robust increases in total STAT1 and STAT3 proteins.
CNTF-mediated activation of MAPK occurs after STAT3 activation in
retina. Cytokine-induced stimulation of MAPK is proposed to occur via
recruitment of Src homology 2 phosphatase (SHP2) to
tyrosine-phosphorylated gp130, interaction between SHP-2 and membrane-associated Ras, and subsequent stimulation of ras-MAPK signaling (Chin et al., 1997; Kim and Baumann, 1999). Activation of
MAPK by CNTF treatment has also been observed in neural precursors in vitro in which it is proposed to also influence GFAP
expression via STAT-dependent and STAT-independent mechanisms (Bonni et
al., 1997; Rajan and McKay, 1998). MAPK activation can downregulate STAT3-mediated transcription by blocking tyrosine phosphorylation of
STAT3 and by phosphorylating STAT3 at serine residues (Jain et al.,
1998; Sengupta et al., 1998). Previous work has shown that the ras-MAPK
pathway is activated in Müller cells by exposure to bright light
and administration of xylazine (Liu et al., 1998; Peng et al., 1998).
Our results suggest that Jak-STAT signaling in retinal glial cells can
be influenced by cross-talk with the ras-MAPK pathway and that both
pathways are likely involved in mediating the effects of CNTF in
promoting neuronal survival in the retina and elsewhere.
A link between CNTF, Jak-STAT, GFAP, and neuroprotection
in vivo
Cytosolic CNTF is released into the extracellular environment
after trauma (Friedman et al., 1992; Sendtner et al., 1992; Stahl and
Yancopoulos, 1994). In adult rat retina, CNTF is localized along
Müller cell processes (Kirsch et al., 1997). Therefore, CNTF
release from damaged Müller cells after local physical trauma may
be responsible for STAT3 activation in nearby, intact Müller cells. However, it is unknown whether CNTF is also released and therefore involved in the activation of STAT3 after conditioning exposure to bright light or stimulation of
2-adrenergic receptors by xylazine. Recent
work has implicated the involvement of Jak-STAT signaling in multiple
physiological processes in the nervous system (Bonni et al., 1997;
Rajan and McKay, 1998). In vitro studies have shown that
STAT3 activation permits astrocytic differentiation from precursor
cells, induces neurite outgrowth in PC12 cells, and produces dendritic
retraction in cultured sympathetic neurons (Wu and Bradshaw, 1996;
Ihara et al., 1997; Guo et al., 1999). STAT3 is phosphorylated in
vivo in neurons in the rat superior cervical ganglion after
transection of the postganglion nerves, in microglia after transient
focal cerebral ischemia, and in cortical glia after excitotoxic lesions
(Rajan et al., 1995; Planas et al., 1996; Acarin et al., 1998). These
findings suggest a role for STAT3 in response to neuronal stress.
Whether STAT3 activation in response to stress is sufficient to promote
neuronal survival is unknown, but the present study demonstrates that
exogenously administered CNTF can directly activate STAT3 in
vivo. Given the documented neuroprotective effects of CNTF in the
CNS and peripheral nervous system, our findings support the direct
involvement of the Jak-STAT pathway in mediating survival of mature neurons.
CNTF-mediated activation of STAT1 and STAT3 can specify cortical
precursors and neuroepithelial stem cells in vitro to adopt an astrocyte phenotype by binding to the gfap promoter and
stimulating GFAP expression (Bonni et al., 1997; Rajan and McKay,
1998). Exogenously administered CNTF and a wide variety of
stress-mediated stimuli induce dramatic upregulation of GFAP in CNS
astrocytes and retinal Müller cells (Eisenfeld et al., 1984;
Guerin et al., 1990; de Raad et al., 1996; Chu et al., 1998; Milam et
al., 1998). Our results are the first to show a direct link between
CNTF-mediated early activation of STAT3 in vivo and the
subsequent upregulation of GFAP in the same glial cells. These findings
indicate that CNTF can directly act on glia, despite the fact that they
appear to express little or no CNTFR mRNA or protein in the intact
retina and CNS (Ip et al., 1993; MacLennan et al., 1996; Kirsch et al., 1997; Fuhrmann et al., 1998).
Given the early STAT3 responsiveness of retinal ganglion cells to CNTF,
activation of the Jak-STAT signaling pathway may directly mediate the
neuroprotective effects of CNTF on ganglion cells (Unoki and LaVail,
1994; Cui et al., 1999). However, CNTF did not activate STAT3 in
photoreceptors. In this context, it is interesting to note that CNTF or
Axokine must be administered 24-48 hr before exposure to toxic levels
of light to maximally protect photoreceptors from damage (M. LaVail,
personal communications). This temporal lag coincides closely
with the Axokine-induced upregulation of GFAP in Müller cells and
the delayed upregulation of total STAT1 and STAT3 levels. Recent work
has shown that the presence of GFAP-positive, "reactive" astrocytes
is associated with protection of neurons from extensive degeneration
caused by localized stab-induced injury (Bush et al., 1999). It is
unknown how "activated" astrocytes support neuronal survival. Our
present results strongly suggest that CNTF-mediated changes in
Müller cell function initiate a secondary, neuroprotective
signaling event to photoreceptors, the nature of which remains to be elucidated.
| |
FOOTNOTES |
Received Nov. 5, 1999; revised March 6, 2000; accepted March 17, 2000.
We thank Drs. George Yancopoulos and John Rudge for their assistance
and guidance in the experimentation, and Evan Burrows and Claudia
Murphy for contributing their expertise to the design of the figures.
We also acknowledge the Regeneron community for supporting the present study.
Correspondence should be addressed to Ward Peterson or Stanley Wiegand,
Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY
10591. E-mail: stan.wiegand{at}regpha.com.
| |
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TOP
ABSTRACT
INTRODUCTION
