Growth Arrest-Specific Gene 6 (Gas6)/Adhesion Related Kinase (Ark) Signaling Promotes Gonadotropin-Releasing Hormone Neuronal Survival via Extracellular Signal-Regulated Kinase (ERK) and Akt
Melissa P. Allen,
Chan Zeng,
Kristina Schneider,
Xiaoyan Xiong,
Mary Kay Meintzer,
Paola Bellosta,
Claudio Basilico,
Brian Varnum,
Kim A. Heidenreich and
Margaret E. Wierman
Research Service (M.P.A., C.Z., K.S., X.X., M.K.M., K.A.H.,
M.E.W.) Veterans Affairs Medical Center and
Departments of Medicine and Pharmacology University of Colorado
School of Medicine Denver, Colorado 80220
Department of
Microbiology and the Kaplan Cancer Center (P.B., C.P.) New
York University School of Medicine New York, New York
10016
Amgen (B.V.) Thousand Oaks, California
91320
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ABSTRACT
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We identified Ark, the mouse homolog of the
receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel
factors involved in GnRH neuronal migration by using
differential-display PCR on cell lines derived at two windows during
GnRH neuronal development. Ark is expressed in Gn10 GnRH cells,
developed from a tumor in the olfactory area when GnRH neurons are
migrating, but not in GT17 cells, derived from a tumor in the
forebrain when GnRH neurons are postmigratory. Since Ark (Axl)
signaling protects from programmed cell death in fibroblasts, we
hypothesized that it may play an antiapoptotic role in GnRH neurons.
Gn10 (Ark positive) GnRH cells were more resistant to serum
withdrawal-induced apoptosis than GT17 (Ark negative) cells, and this
effect was augmented with the addition of Gas6, the Ark (Axl) ligand.
Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and
the serine-threonine kinase, Akt, a downstream component of the
phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or
Akt activation is required for the antiapoptotic effects of Gas6/Ark in
GnRH neurons, cells were serum starved in the absence or presence of
Gas6, with or without inhibitors of ERK and PI3-K signaling cascades.
Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by
coincubation of the cells with the mitogen-activated protein/ERK kinase
(MEK) inhibitor, PD98059, or wortmannin (but not rapamycin).
These data support an important role for Gas6/Ark signaling via the ERK
and PI3-K (via Akt) pathways in the protection of GnRH neurons from
programmed cell death across neuronal migration.
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INTRODUCTION
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GnRH Neurons: A Model of Tightly Controlled Neuronal Migration
across Embryogenesis
GnRH is the hypothalamic releasing hormone that controls pituitary
gonadotropin subunit biosynthesis and, ultimately, reproductive
function (1). The GnRH neuronal population is unique, in that 800-1000
neurons migrate from the olfactory placode to the forebrain across
embryogenesis in the rodent (2, 3, 4, 5). The neurons migrate adjacent to
olfactory neurons, but then diverge to reach their final destination in
the hypothalamus. GnRH neurons innervate the anterior pituitary and
deliver an episodic pattern of hormone signaling to the gonadotropes
that ultimately leads to normal reproductive function (1). Failure of
this targeted GnRH neuronal migration results in failure of sexual
maturation and GnRH deficiency syndromes (6, 7). In the human, the
X-linked form of GnRH deficiency has been found to result from a defect
in the KAL gene, which encodes a neural cell adhesion molecule
expressed in cells adjacent to GnRH neurons (8, 9, 10). The molecular
defects for other forms of GnRH deficiency are unknown due, in part, to
the lack of information concerning the factors that control GnRH
neuronal migration.
GnRH Neuronal Cell Lines: Models to Study GnRH Expression
Our ability to directly study GnRH gene expression was advanced by
the development of GnRH-producing cell lines. Mellon and colleagues
(11) used the rat GnRH promoter fused to the SV40 T antigen
in transgenic animals to target immortalization of the GnRH neuronal
population. One animal developed a tumor in the forebrain, a time when
GnRH neurons are postmigratory. The GT cell lines were derived from
that tumor and produce large amounts of GnRH mRNA and protein (12).
Similarly, Radovick and co-workers (13) used the human GnRH promoter
fused to the SV40 T antigen in transgenic mice. An animal
developed a GnRH neuronal tumor in the olfactory area, a locus for
migrating GnRH neurons. The resultant Gn10, Gn11, and NLT cell lines
express low levels of GnRH mRNA and protein (Ref. 13 , and M. E.
Wierman, unpublished observations). Based on the divergent phenotypes
of the two neuronal cell lines, we hypothesized that by using the
technique of differential display PCR, we could identify novel factors
involved in GnRH neuronal migration and/or gene expression (14). We
reasoned that GT17 cells would express factors resulting in high
level GnRH expression and a postmigratory status. Conversely, Gn10
cells would express factors involved in GnRH neuronal migration and
factors that repress GnRH expression (or lack activators of gene
expression). Gn801 is a cDNA clone characterized in this screen from
Gn10 cells and was identified as Ark, a membrane receptor tyrosine
kinase that may play a role in GnRH neuronal migration, gene
expression, and protection from apoptosis.
Ark (Axl): Role in Protection from Apoptosis
Ark is a mouse protein identified during a screen for homologs of
the BEK fibroblast growth factor receptor (15). Ark and its human
homolog, Axl, Ufo, or Tyro7 (16, 17, 18), are members of a new family of
receptor tyrosine kinases that includes Tyro3 (with many alternative
names) (17, 19) and Mer (20). This family is unique in that the
N-terminal, extracellular portion of the molecule contains two Ig-like
repeats and two fibronectin type III repeats (15, 16, 17, 18, 19, 20). This combination
of structural elements has been classically observed in cell adhesion
molecules or receptor tyrosine phosphatases, but not in receptor
tyrosine kinases. Although initially isolated as candidate growth
factor receptors, members of the Ark (Axl) family are not mitogenic
unless they are overexpressed at high levels in tumor cell lines
(21, 22). Some have suggested the importance of the extracellular
domain of Ark in cell-cell adhesion due to its ability to induce
homophilic binding independent of ligand in fibroblasts (21), although
others have not shown an effect independent of ligand using Axl
expressing 32D cells (23). The nuclear transcription factors and gene
targets downstream in the Ark-signaling pathway have not been
identified.
Gas6/Ark Signaling in Other Systems Protects from Programmed Cell
Death
Recently, the ligand for Ark (Axl) and Tyro3 was identified as
Gas6, a gene induced after growth arrest of fibroblast cells in culture
(24, 25, 26). Gas6 is a soluble, vitamin K-dependent protein with homology
to protein S (24, 25, 26). Studies have suggested the importance of
Gas6/Axl-induced chemotaxis of 32D and vascular smooth muscle cells
(23, 27). Additional studies show that Gas6/Ark or Axl signaling
reduces the rate of programmed cell death in fibroblasts (28, 29). We
were intrigued by the identification of Ark in Gn10 GnRH neuronal
cells, which were derived from migrating GnRH neurons, and hypothesized
that Ark (Axl) may play a role in protection of the GnRH neuronal
population from apoptosis during their migration into the forebrain.
Thus, we designed experiments in the two divergent GnRH-producing cell
lines to address the role of Gas6/Ark (Axl) signaling during neuronal
cell death and the pathways that are activated in this process.
Pathways Involved in Neuronal Apoptosis
The downstream signaling cascades involved in apoptosis are a
focus of active investigation. Many of the pathways are similar among
cell types, but some cell-specific pathways have been identified.
Studies have shown the importance of the extracellular signal-regulated
kinase (ERK) pathway in rescue from cell death (30, 31, 32, 33, 34, 35). It is unclear
however, whether the activation of ERK is to trigger mitogenesis and
thereby indirectly regulate apoptosis or to act directly in this
process (28). Studies have also shown the importance of Akt [also
called protein kinase B (PKB) and related to A and C protein kinase
(RAC-PK)], a downstream component of the phosphoinositide
3-kinase (PI3-K) pathway, in the protection of cells from apoptosis
(36, 37, 38, 39, 40, 41). Recently, investigators have demonstrated that Akt
phosphorylates the BCL-2 member, BAD, deactivating its
proapoptotic actions (42). In addition to antiapoptotic signaling
cascades, stress-activated proapoptotic pathways converge in activation
of p38 mitogen-activated protein (MAP) kinase in neuronal cells (43).
Inhibitors of p38 have been shown to protect from various triggers of
programmed cell death (43). With this background, we examined which of
these pathways may be involved in Gas6/Ark signaling in GnRH neuronal
cells.
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RESULTS
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The Ark Tyrosine Kinase Receptor mRNA and Protein Are Expressed in
Gn10 and Not GT17 Neuronal Cells
To confirm that Ark mRNA and functional Ark protein were expressed
in GnRH neuronal cells from which the clone Gn801 was isolated,
Northern and Western blots from each neuronal cell line were performed.
The full-length mouse Ark cDNA was used to probe the Northern (15). An
Ark-specific antisera, no. 318, which recognizes the Ark extracellular
domain (21), was used to detect the Ark protein. Figure 1
shows the presence of the Ark 3.4-kb
mRNA and the 120140 kDa protein doublet in the Gn10, but not in the
GT17 neuronal cells. [Additional studies showed that neither
neuronal cell line contains detectable levels of Gas6 mRNA or protein
(data not shown).]

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Figure 1. Ark Is Expressed in Gn10 and Not GT17 Neuronal
Cells
Twenty micrograms of total RNA from each neuronal cell line were
separated by electrophoresis on a 1.4% agarose gel, transferred to
nitrocellulose, and hybridized with a radiolabeled Ark cDNA. The 3.4-kb
mRNA is Ark. Total cell lysates of the two neuronal cells were
separated by electrophoresis on a 10% SDS-PAGE gel, transferred to
PVDF, and probed with the No. 318 Ark antisera. The
arrow indicates the 120- to 140-kDa Ark protein.
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Ark-Positive Gn10 Neurons Are More Resistant to Growth Factor
Withdrawal- Induced Apoptosis than Ark-Negative GT17 Cells
Prior experiments in fibroblast cell lines demonstrated that
Gas6/Ark (Axl) signaling is important in protecting or rescuing cells
from programmed cell death induced by serum withdrawal (28, 29). This
finding prompted us to determine whether a similar function is
important in GnRH neuronal cells. If Ark expression and activation
protect neurons from apoptosis, then the Gn10 (Ark positive) cells
might be more resistant than GT17 (Ark negative) neurons to growth
factor withdrawal, a standard paradigm to trigger apoptosis. To test
this hypothesis, the GT17 and Gn10 cells were grown under normal
conditions and then changed to serum-free media. Various times after
serum withdrawal (272 h), cells were stained for condensed,
hyperchromatic nuclei using the Hoescht stain (33258). Gn10 GnRH
neurons were less sensitive to serum withdrawal than GT17 cells. At
baseline, they exhibited a low rate of apoptosis (2.2%) that did not
increase over the first 24 h and then gradually increased to 7.3%
and 12.4% at 48 and 72 h, respectively. In contrast, GT17 GnRH
neurons were more sensitive to growth factor removal. They showed a
basal level of apoptosis of 2.9%. This increased to 13.8%, 42.3%,
and 41.2% at 24 h, 36 h, and 48 h, respectively. When
serum was removed, both neuronal cells lost their ability to
remain attached to the tissue culture dishes, which was partially
prevented by culturing on polylysine-coated slides.
To further map the time course of sensitivity to serum
withdrawal, both neuronal cells were placed in serum-free conditions,
and cells were stained at various time intervals with acridine
orange/ethidium bromide to detect apoptotic nuclei as orange-red
condensed fragments (Fig. 2
). In this
assay, cells are lifted, thereby capturing both attached and detached
cells. In GT17 cells, the basal level of apoptosis in serum was 2.1%
and was similar at 3% at 8 h after serum withdrawal, but then
increased to 28%, 35%, and 38% at 24 h, 48 h, and 72
h. In contrast in Gn10 neuronal cells, the basal rate of apoptosis was
2% and was stable over the first 24 h of serum withdrawal (2.1%
and 2.5%). The levels of apoptosis then increased gradually over the
next 2 days to 10.6% and 13% at 48 and 72 h (Fig. 2
). Thus,
there was a clear dichotomy between Ark-positive Gn10 and Ark-negative
GT17 cells in apoptotic rates after serum deprivation. We hypothesize
that Ark is one of many factors contributing to the differences between
the two GnRH neuronal cell lines that may play a role in the regulation
of programmed cell death in GnRH neurons.

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Figure 2. Gn10 Cells Are More Resistant to Serum
Deprivation-Induced Programmed Cell Death Than GT17 GnRH Neuronal
Cells
Numbers of apoptotic cells were determined by ethidium bromide/acridine
orange staining at various times after cells were placed in serum-free
media.
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Gas6/Ark Activation Triggers Multiple Signaling Cascades in GnRH
Neuronal Cells
To explore the intracellular pathways by which Gas6/Ark might
modulate the sensitivity to apoptosis in GnRH neurons, we asked whether
rhGas6 triggered changes in the activation of ERK and Akt. ERK1 and
ERK2 are components of the MAP kinase pathway known to be critical in
mitogenic as well as antiapoptotic signaling in certain cell systems
(30, 31, 44). Akt is a downstream component of the PI3-K pathway, known
to be activated by growth factors and to protect cells from apoptotic
stimuli (30, 36, 38, 39, 40, 41, 45). Phosphorylation of these proteins, as
detected by phospho antisera, is correlated with their activation.
GT17 and Gn10 cells were grown in the presence or absence of
serum (48 h), and Gn10 cells were stimulated with rhGas6, 400 ng/ml,
for various periods of time. Whole-cell lysates were analyzed by
Western blot and probed with antisera specific for phospho-ERK and ERK
(Fig. 3A
) or phospho-Akt and Akt (Fig. 3B
). Of interest, GT17, Ark-negative, cells grown in serum
had lower levels of activated ERK and Akt than Gn10, Ark-containing
neurons, and levels did not change with serum withdrawal (lanes 1 and 2
vs. lanes 3 and 4, panels A and B). However, in the presence
of serum, Gn10 neuronal cells have relatively high levels of active ERK
and Akt (lanes 3 and 5, panels A and B). Serum withdrawal decreased
levels of phospho-ERK and Akt in the Gn10 cells with more dramatic
effects on ERK. In Gn10 cells, addition of rhGas6 under serum-free
conditions stimulated ERK (13-fold) and Akt (1.5-fold)
(P < 0.05) activity with no significant effect on ERK
or Akt levels. The activation of ERK was rapid and returned to a
baseline higher than control by 60 min. The activation of Akt was rapid
but transient, reaching a maximum at 5 min and returning to basal by 20
min.

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Figure 3. Gas6/Ark Activation Is Associated with Activation
of ERK and Akt
GT17 and Gn10 cells were grown in serum or serum starved for 48
h (left), and Gn10 cells were then treated with vehicle
or rhGas6 (400 ng/ml) for 160 min (right). Cell
lysates were separated by electrophoresis, transferred to PVDF, and
incubated with antisera specific for phospho-ERK, ERK (panel A), or
phospho-Akt and Akt (panel B). Results are shown from a representative
experiment from three to five performed. Panel C shows phosphorylation
of histone H2B by Akt in response to rhGas6 (400 ng/ml) for 120
min.
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To confirm the increases in levels of phospho-Akt by Gas6, an
alternative assessment of Akt activity was made by testing its ability
to phosphorylate the substrate histone H2B in an immunocomplex kinase
assay (40). Gn-10 cell lysates were harvested after exposure to serum
or after serum withdrawal in the absence or presence of rhGas6. Figure 3C
shows a rapid 2.3-fold increase in the phosphorylation of histone
H2B by Akt at 5 min after addition of rhGas6 with a rapid return to
basal levels. These data confirm that Gas6/Ark (Axl) signals through
Akt in Gn10 cells.
The activity of p38 MAP kinase was also assessed in the neuronal cells,
as it has recently been shown to trigger neuronal apoptosis (43) and
was expected to be inhibited by Gas6 (Fig. 4
). The activity of p38 MAP kinase was
undetectable in Gn10 neurons after 48 h of serum deprivation in
the absence or presence of rhGas6. This lack of activity was
observed despite fairly high basal concentrations of the enzyme (Fig. 4
). Together, these data suggest the potential importance of the ERK
and PI3-K pathways and not p38 MAP kinase to transmit the Ark (Axl)
signal in GnRH neuronal cells.

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Figure 4. Serum Deprivation and Gas6 Do Not Activate p38 MAP
Kinase in Gn10 Neuronal Cells
Gn10 cells were grown in serum-free media for 48 h and then
treated with vehicle or rhGas6 (400 ng/ml) for 10 min. Cell lysates
were separated by electrophoresis, transferred to PVDF, and incubated
with antisera specific for phospho-p38 MAP kinase and p38 MAP kinase.
Lane 5 contains active p38 as a control.
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The Mitogen-Activated Protein/ERK Kinase (MEK) Inhibitor,
PD98059, Blocks ERK Activation by Gas6
To test the hypothesis that activation of the ERK pathway is
involved in Gas6/Ark rescue from neuronal programmed cell death, Gn10
cells were grown in serum or in serum-free media for 48 h. Cells
were treated with vehicle, rhGas6 (400 ng/ml, 5 min), or IGF-I (100
ng/ml, 15 min). Some cells were preincubated with the MEK inhibitor,
PD98059, which blocks upstream of ERK (31, 43), for 1 h before the
addition of rhGas6. Cell lysates were analyzed by Western blot with ERK
and phospho-ERK antibodies. Serum deprivation decreased the levels of
activated ERK in the neuronal cells (Fig. 5
, lanes 1 and 2), while rhGas6 triggered
a 10- to 20-fold increase in levels of phospho-ERK at 5 min (lane 3).
The stimulation of ERK by rhGas6 was greater than that achieved by
addition of IGF-I in these neuronal cells (IGF-I, 2-fold stimulation,
lane 4). The activation of ERK by rhGas6 or IGF-I was blocked in the
presence of the MEK inhibitor, PD98059, to below that in unstimulated
cells (lanes 6 and 7). There were no changes observed in total ERK
levels after rhGas6 addition.

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Figure 5. The MEK Inhibitor, PD98059, Blocks ERK Activation
by Gas6
Gn10 cells were serum starved for 48 h and then treated with
rhGas6 (400 ng/ml) for 5 min or IGF-I (100 ng/ml) for 15 min. In some
cases, the cells were pretreated with PD98059 (20 µM) for
1 h. Cell lysates were separated by electrophoresis, transferred
to PVDF, and incubated with antisera specific for phospho-ERK and ERK.
Lane 1, Gn10 cells grown in serum; lane 2, serum starved; lane 3, with
rhGas6; lane 4, with IGF-I; lane 5, with PD98059; lane 6, with rhGas6
and PD98059; lane 7, with IGF-I and PD98059.
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Inhibitors of the ERK Pathway Block Gas6/Ark Protection from
Neuronal Apoptosis
To investigate the importance of the ERK-signaling pathway in
Gas6/Ark protection of GnRH neuronal cells during serum
withdrawal-induced cell death, Gn10 cells were grown in serum-free
media in the presence or absence of rhGas6 (400 ng/ml) with or without
the MEK inhibitor, PD98059 (Fig. 6
).
Serum withdrawal for 48 h resulted in an increase in apoptosis as
measured by Hoescht staining from 2.3% to 12.3% (lanes 1 and 2).
rhGas6 protected Gn10 cells from programmed cell death, decreasing
levels to 4.8% (lane 3). Incubation of cells with the MEK inhibitor
alone had no effect on serum withdrawal induced apoptosis (lane 4);
however, Gas6 protection was lost in the presence of the MEK inhibitor
(lane 5). As another control, there was no effect of
bisindolymaleimide, an inhibitor of the protein kinase C pathway (46),
on Gas6 rescue of GnRH neuronal cells from growth factor
withdrawal-induced cell death (lane 6). These results show the
importance of the ERK pathway in Gas6/Ark (Axl) antiapoptotic signaling
in GnRH neuronal cells.

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Figure 6. Inhibition of the ERK Pathway Blocks Gas6 Rescue of
Gn10 Neuronal Cells from Apoptosis
Gn10 cells were grown in serum-free media for 48 h in the absence
or presence of rhGas6 (400 ng/ml) and/or the MEK inhibitor, PD98059 (20
µM), or bisindolymaleimide (100 nM).
Apoptotic cells were counted by Hoescht staining. Results are the
mean ± SEM of three experiments. Lane 1,
Baseline apoptosis; lane 2, 48 h of serum deprivation; lane 3,
with rhGas6; lane 4, with PD98059; lane 5, with rhGas6 and PD98059;
lane 6, with rhGas6 and bisindolymaleimide.
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Recent studies suggested that ERK activation by Gas6/Ark involves
induction of mitogenesis that may indirectly contribute to protection
from apoptosis (28). Therefore, Gn10 neuronal cells were grown in the
presence or absence of Gas6, and cell counts were determined. There
were no significant mitogenic effects of Gas6 in these neuronal cells
as assessed by cell counts (data not shown). Additional studies were
performed using BrdU incorporation in the absence or presence of Gas6.
Cells were serum starved for 24 h and incubated in the presence or
absence of rhGas6 (400 ng/ml) for 24 h. These studies also showed
no effect of rhGas6 on Gn10 neuronal cells to augment BrdU
incorporation (rhGas6 69.8 ± 0.13% vs. serum-starved
control cells 100 ± 0.12%). Together, these experiments suggest
that activation of the ERK pathway during Gas6/Ark rescue from neuronal
apoptosis is not dependent on a mitogenic signal to trigger entry into
the cell cycle and support the direct role of the pathway in protection
from programmed cell death.
Wortmannin Blocks Akt Activation by Gas6
To test whether Akt is also important in Gas6/Ark protection of
GnRH neurons from apoptosis, Gn10 neuronal cells were grown in
serum-free media for 48 h and then treated with vehicle, rhGas6,
or IGF-I as a control growth factor stimulus. Some cells were
preincubated with wortmannin (1 µM) to inhibit the PI3-K
pathway upstream of Akt (4). Cell lysates were analyzed by Western blot
with phospho-Akt and Akt antisera (Fig. 7
). Growth factor withdrawal decreased,
but did not abolish, activated Akt levels (lower band, lanes
1 and 2). rhGas6 addition, however, consistently increased the levels
of phospho-Akt by 5 min (lower band, lane 3). The
stimulation of Akt by rhGas6 was comparable to the stimulation of Akt
by IGF-I (lower band, lane 4). Wortmannin completely blocked
the stimulation of Akt by both rhGas6 and IGF-I (lanes 6 and 7). In
addition, wortmannin lowered phospho-Akt levels to below baseline,
again supporting the importance of other factors in Gn10 cells that
sustain an activated PI3-K via Akt signal. No changes in the protein
levels of Akt were observed in the various experimental
manipulations.

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Figure 7. Wortmannin Blocks Akt Activation by Gas6
Gn10 cells were serum starved for 48 h and treated with vehicle or
rhGas6 (400 ng/ml) for 5 min or IGF-I (100 ng/ml) for 15 min in the
absence or presence of pretreatment with wortmannin (1
µM) for 20 min. Cell lysates were separated by
electrophoresis, transferred to PVDF, and incubated with phospho-Akt
and Akt antisera. Lane 1, Gn10 cells grown in serum; lane 2, serum
starved; lane 3, with rhGas6; lane 4, with IGF-I; lane 5, with
wortmannin; lane 6, with rhGas6 and wortmannin; lane 7, with IGF-I and
wortmannin.
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Inhibition of the PI3-K Pathway Blocks Gas6/Ark Rescue of Gn10 GnRH
Neurons from Serum Withdrawal-Induced Apoptosis
To confirm that the changes observed in Akt activation were
directly relevant to Gas6/Ark protection from neuronal apoptosis, Gn10
neurons were grown in serum-free media in the absence or presence of
wortmannin (100 nM), rapamycin (20 ng/ml), rhGas6 (400
ng/ml), or combinations of the above (Fig. 8
). Gn10 cells exposed to growth factor
withdrawal for 48 h had an increased rate of apoptosis as
determined by Hoescht staining from 2.2% to 11.7% (lanes 1 and
2). Incubation with rhGas6 partially rescued the Gn10 neurons from
programmed cell death (5.4% at 48 h, lane 3). Incubation of
neuronal cells with wortmannin resulted in higher levels of apoptosis
(20.9%, lane 4) compared with serum withdrawal (11.7%). These results
suggest the PI3-K pathway is used by other membrane receptors to
protect GnRH neurons from cell death in addition to those controlling
apoptosis due to growth factor deprivation. rhGas6 was unable to
protect the neuronal cells in the presence of the wortmannin blockade
of the PI3-K pathway (lane 5). There was no effect of rapamycin, a
selective inhibitor of S6 kinase that is also downstream of PI3-K (47),
on the rates of apoptosis (lane 6). In addition, rapamycin did not
influence the ability of rhGas6 to rescue from neuronal
apoptosis (lane 7), suggesting that Akt and not S6 kinase is the
important downstream target of PI3-K in these cells. Together, these
data support the hypothesis that Gas6/Ark protection of GnRH neurons is
mediated through mechanisms that involve Akt as well as ERK.

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Figure 8. Inhibition of the PI3-K Pathway Blocks Gas6 Rescue
of Gn10 Neurons from Serum Withdrawal-Induced Apoptosis
Gn10 neurons were grown in serum-free media for 48 h in the
absence or presence of rhGas6 (400 ng/ml), wortmannin (100
nM), rapamycin (20 ng/ml), or combinations of the above.
Cells were harvested and rates of apoptosis determined by staining with
Hoescht 33258. Results are the mean ± SEM of three
experiments. Lane 1, Baseline apoptotic cells in Gn10 cells; lane 2,
48 h in serum-free media; lane 3, with rhGas6; lane 4, with
wortmannin; lane 5, with rhGas6 and wortmannin; lane 6, with rapamycin;
lane 7, with rhGas6 and rapamycin.
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DISCUSSION
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Programmed Cell Death in GnRH Neurons
During development, apoptosis plays a critical role in determining
the ultimate fate of neuronal populations. Studies have estimated that
2080% of the neurons expressed during embryogenesis die before
maturation of the organism (34). The GnRH neuronal population is unique
in that, unlike other neurons that express hypothalamic releasing
factors, the neurons must migrate from the olfactory placode into the
forebrain. In addition, the population is small, with only 8001000
neurons in the rodent making the journey (2, 3, 4, 5). Because of the
ultimate importance of appropriately targeting this subpopulation of
neurons into the hypothalamus for reproductive competence, one might
expect a complex series of mechanisms to modulate the rate of
programmed cell death during their migratory journey. The ability to
identify factors involved in this process has been limited by the small
size of the neuronal population and difficulty in working with primary
cultures. We took advantage of two available GnRH-producing neuronal
cell lines to use differential display-PCR to clone candidate
cDNAs that might play a role in GnRH neuronal migration or gene
expression (14). Ark, expressed in Gn10 GnRH cells derived when GnRH
neurons are migrating, is the first candidate to be studied
functionally. The potential role of Ark (Axl) in cell-cell contact has
been suggested in 32D myeloid cells (23, 24, 25, 26, 27) and in vascular smooth
muscle cells (27), but it has not been studied in neuronal cells. The
role of Ark (Axl) in the modulation of GnRH gene expression is under
active investigation in the laboratory (48). The present study strongly
supports Arks role in protection of the GnRH neuronal population from
programmed cell death.
Since Ark is not expressed in the GT17 cells derived from GnRH
neurons in the forebrain, one might expect that additional protective
mechanisms against apoptosis of the GnRH population are not needed once
the appropriate targeting has taken place. Gas6 is not expressed in
these neuronal cells, so one would hypothesize that adjacent glia or
neuronal cells synthesize the Ark (Axl) ligand during the
migratory process. This is consistent with an increasing literature
supporting the role of glial elements in neuronal migration and
survival (49).
The Functional Role of Ark to Protect from Programmed Cell
Death
Serum deprivation is a classic model system to study programmed
cell death. Although most studies of the Ark (Axl) family have been
performed in this model system, Bellosta et al. (21)
recently reported that Gas6/Ark signaling protects fibroblasts from
apoptosis induced by tumor necrosis factor-
and c-Myc but not
that induced by UV irradiation or staurosporine. Similar studies of Ark
function in neuronal cells using these alternative model systems of
apoptosis have not yet been performed. To support the physiological
importance of Ark (Axl), however, embryonic fibroblasts from Ark (Axl)
knockout mice were found to be more susceptible to growth factor
withdrawal-induced apoptosis than wild-type cells (21). Together, these
data suggest the general role of Ark (Axl) in modulating the rate of
programmed cell death.
Gas6/Ark Signaling in Neuronal Cells Is Different Than in Other
Systems
Ark was initially derived in studies searching for novel growth
factor receptors, and Axl has been found to be overexpressed in some
leukemia cell lines (18, 27). Initial studies focused on the role of
the kinase as a mitogenic stimulus, but recent studies have suggested
Ark (Axl) is only a weak mitogen unless overexpressed (22, 28). In GnRH
neuronal cells, Gas6/Ark (Axl) signaling is not associated with a
mitogenic response, which is expected since neuronal cells are not
subject to significant proliferative responses.
In the GnRH neuronal cell lines, both the ERK and Akt pathways were
activated in the presence of serum. The Gn-10 (Ark positive) cells,
however, had higher levels of activated ERK and Akt than the GT17
(Ark negative) cells, suggesting that the high endogenous levels were
not due solely to SV40 TAg immortalization. In addition,
growth factor withdrawal decreased activated ERK levels dramatically
with less effect on activated Akt levels, supporting the recent
observations that many neuronal cells use the PI3-K via Akt pathway as
the major control point for multiple endogenous signals that mediate
neuronal survival (38, 40, 45).
The ERK intracellular signaling system has been associated with both
proapoptotic and antiapoptotic effects (reviewed in Ref. 32). Gardner
and Johnson (33) demonstrated that fibroblast growth factor 2
suppression of tumor necrosis factor-
mediated apoptosis required
Ras activation of ERK in L929 cells. Párrizas et al.
(44) showed the activation of both ERK and PI3-K pathways in
IGF-I-mediated rescue from apoptosis in differentiated PC12 cells (44).
Bellosta and co-workers (21) have shown in NIH-3T3 cells that Ark
triggered a modest increase in ERK activity that accompanied the
increased survival of cells at concentrations that did not promote DNA
synthesis. In our studies, incubation with the MEK inhibitor (PD98059)
completely reversed Gas6 protection from programmed cell death in GnRH
neuronal cells. These data show the critical role of the ERK pathway in
transmitting the Gas6 signal from the membrane to the appropriate
intracellular targets to rescue these neuronal cells from growth factor
withdrawal-induced apoptosis. In contrast to Gas6/Ark, IGF-I triggered
activation of Akt to a greater extent than ERK. These results confirm
the cell specificity of intracellular signaling pathways downstream of
growth factor receptors.
In prior studies of other downstream signaling pathways activated by
Gas6/Ark (Axl), divergent results have been reported (22, 28, 29).
Goruppi and co-workers (29) have suggested the importance of PI3-K
working through S6K as well as Src activation in both mitogenic and
survival activities by Gas6/Axl signaling in NIH-3T3 cells. The data
concerning Src activation are complex since the Axl cytoplasmic domain
lacks Src consensus binding sequences, and Src could not be
coimmunoprecipitated with the receptor (29). Since the NIH-3T3 cells
contain Tyro3, another family member that heterodimerizes with Ark
(Axl), the complement of protein partners in each cell may influence
the importance of different signaling pathways. However, the GnRH
neuronal cells do not express Tyro3 (X. Xiong and M. E. Wierman,
unpublished observations), suggesting the observed responses are due
solely to Ark (Axl) activation.
Goruppi and colleagues (29) also found that rapamycin treatment
inhibited the ability of Gas6 to prevent apoptosis-associated Gas2
proteolytic cleavage. In our studies, rapamycin had no effect on Gas6
protection from apoptosis, in contrast to wortmannin. Together with the
data showing increased phospho-Akt and phosphorylation of the Akt
substrate, histone H2B, by Gas6, these results suggest that in GnRH
neurons, the PI3-K pathway triggers Akt as an additional downstream
effector to modulate the sensitivity of GnRH neurons to trophic factor
withdrawal. The recent demonstration that Akt inhibits apoptosis in
cerebellar neurons (40), in rat hippocamal H197 neuronal cells (45),
and in sympathetic neurons (38) and that Akt also phosphorylates the
proapoptotic BCL-2 member, BAD, in neuronal cells to inactivate
it (42) are all supportive of the major importance of this pathway in
mediating multiple receptor-mediated antiapoptotic signals. Since
blockade of either the PI3-K or the ERK pathways reversed the
protective effect of Gas6, it is unclear whether these are parallel
independent signaling systems or whether there is cross-talk at some
level between the components. Future studies will be needed to
investigate these possibilities.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
GT17 and Gn10 GnRH neuronal cells were grown in DMEM
supplemented with 5% FCS, penicillin (100 U/ml), and streptomycin (100
µg/ml). For serum starvation, cells were plated at 60% confluency,
grown overnight, and changed to serum-free media, and the incubation
was continued for 672 h depending on the experimental design.
Reagents
Wortmannin [a specific inhibitor of PI3-K (50)], rapamycin [a
specific inhibitor of p70 S6 kinase (47)], bisindolymaleimide [a
specific inhibitor of protein kinase C (46)], and insulin-like growth
factor 1 (IGF-I) were purchased from Sigma Chemical Co. (St. Louis,
MO). PD98059 (an inhibitor of MEK1 and MEK2, the upstream regulators of
ERK) was purchased from New England Biolabs, Inc. (Beverly, MA)
Purified IgG Ark#318 was raised against the Ark extracellular domain
(21). Antibodies specific to Akt and phospho-Akt were purchased from
New England Biolabs, Inc. Antiactive MAP kinase pAb
(phospho-ERK) and anti-active p38 antibodies were purchased from
Promega, Inc. (Madison, WI). Antisera specific to ERK2 and p38 MAP
kinase were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Recombinant human Gas6 (rhGas6) from Amgen (Thousand Oaks, CA) was
used in all studies (26).
Quantification of Apoptotic Cells
Neuronal cells were grown in standard conditions with or without
FCS for various time intervals. For Hoescht staining, cells were fixed
on dishes or polylysine-coated slides in 1% paraformaldehyde for 2 min
at room temperature followed by 70% ethanol in glycine buffer (100
mM, pH 3.0) for 20 min at -20 C. After fixation, the cells
were washed with PBS three times and then incubated in Hoescht 33258
stain (8 µg/ml in PBS) for 15 min at room temperature. The cells were
washed with PBS three times and then stored in the dark immersed in
PBS. The stained cells were viewed under a fluorescent microscope
(Olympus IMT-2 inverted microscope, Olympus Corp., Lake Success,
NY). Apoptotic cells were measured by counting the number of
neuronal cells with condensed or fragmented chromatin. These cells
typically appeared small and rounded and bright green. One thousand
cells were counted from eight randomly chosen fields. The rate of
apoptosis was expressed as a percentage of total counted cells.
For ethidium bromide/acridine orange staining, cells (floating and
attached) were resuspended in cold PBS. A 1:2 dilution of 100 µg/ml
solutions of ethidium bromide and acridine orange was mixed with the
resuspended cells. The stained cells were viewed as above. Apoptotic
cells were scored as those with orange-red fragmented, condensed
nuclei. The rate of apoptosis was expressed as a percentage of total
counted cells.
Western Blot Analysis
Three million GT17 or Gn10 neuronal cells were grown for
48 h in standard media with or without serum. After the
experimental manipulation, cells were washed twice with PBS (4 C), and
lysed in 250500 µl of cell lysis buffer containing 10
mM Tris-Cl, pH 7.5, 150 mM NaCl, 1
mM EDTA, 1 mM EGTA, 0.1% NP-40, 1% glycerol,
1 mM dithiothreitol, one protease inhibitor tablet/50 ml
(Boehringer Mannheim, Indianapolis, IN), and freshly added 20
mM Na3VO4, 25 mM NaF,
and 20 mM sodium pyrophosphate at 4 C. Lysed cells were
sonicated (Branson sonifier 250, at power 3, Branson Sonic Power Co.,
Danbury, CT) for 10 pulses. The lysate was then spun at 14,000 x
g for 10 min at 4 C. The supernatant was then assayed for
protein concentration with the BCA protein assay kit (Pierce, Inc.,
Rockford, IL). An aliquot of 2030 µg of total protein was resolved
by SDS/PAGE on 1012% gels using a Bio-Rad mini-gel system (Bio-Rad
Laboratories, Richmond, CA). Subsequently, proteins were transferred to
Hybond polyvinylidene difluoride (PVDF) (Amersham Lifesciences,
Inc., Arlington Heights, IL) at 100 V for 1 h at 4 C. The
membranes were blocked in 5% milk, TBS-T buffer (20 mM
Tris-Cl, pH 7.6, 137 mM NaCl, 0.1% Tween-20) overnight at
room temperature. Protein present on the blot was visualized using
enhanced chemiluminescence (ECL) immunodetection reagents (Amersham
Life Sciences, Inc.). For each antiserum, the primary antibody was
diluted to 1:5001:2000 and incubated with the membrane at room
temperature for 2 h. The secondary antibody was diluted
1:20001:3000 and incubated with the membrane for 1 h at room
temperature. An additional four washes were performed before
immunodetection according to the manufacturers instruction (Amersham
Lifesciences, Inc.).
Immune-Complex Kinase Assay for Akt Activity
Gn10 cells were growth for 48 h in normal growth media or
serum-free media. The serum-starved cells were incubated with 400 ng/ml
rhGas6 for various times, washed once with PBS (4 C), and lysed in 137
mM NaCl, 10% glycerol, 1% NP-40, 20 mM Tris,
pH 8, one protease inhibitor tablet/50 ml, 20 mM
Na3VO4, 25 mM NaF, and 20
mM sodium pyrophosphate at 4 C. The lysed cells were
sonicated and spun as described above. Total protein (25 mg) was
immunoprecipitated with 10 µg of antihuman Akt-1 pleckstrin
homology domain antibody (Upstate Biotechnology, Lake Placid, NY) for
1.5 h at 4 C with constant mixing. Subsequently, 100 µl of
protein A/G agarose (Calbiochem, La Jolla, CA) was added and the
incubation continued for 1 h. The immune complexes were washed
three times in lysis buffer, once in dH2O, and once in
kinase assay buffer minus ATP (20 mM HEPES, pH 7.4, 10
mM MgCl2, 10 mM MnCl2)
at 4 C. Akt kinase activity was determined by incubating the immune
complexes with 2 µg histone H2B, 10 µCi [
32P]ATP,
5 µM ATP, and kinase assay buffer for 30 min at room
temperature. The supernatants were resolved by 15% SDS-PAGE, and the
phosphorylation of histone H2B was detected by autoradiography.
Northern Analyses
Twenty micrograms of total RNA isolated from GT17 and Gn10
neuronal cells were separated by electrophoresis in a 1.4%
formaldehyde agarose gel transferred to nitrocellulose and baked at
-80 C as previously described (14). The blot was hybridized with
32P-radiolabeled Ark cDNA (15), washed, and exposed to film
at -70 C for 12 days.
BrdU Labeling for Cell Proliferation
Gn10 neuronal cells were grown in serum-free media for 24 h
and then incubated with vehicle or rhGas6 (400 ng/ml) for 24 h.
Cells were then labeled with BrdU for 24 h using the cell
proliferation enzyme-linked immunosorbent assay (ELISA) colorometric
kit from Boehringer Mannheim. BrdU incorporation was assessed by
colorometric assay using a Biotek ELISA reader. Data represent the
mean ± SEM of six to eight dishes.
 |
ACKNOWLEDGMENTS
|
---|
We acknowledge the excellent secretarial support of Ms. Gloria
Smith.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Margaret E. Wierman, Endocrinology (111H), Veterans Affairs Medical Center, 1055 Clermont Street, Denver, Colorado 80220. E-mail: margaret.wierman{at}uchsc.edu
This work was supported by NIH Grant HD-31191 (to M.E.W.). Dr. Wierman
has a Career Development Award from the Veterans Administration.
Received for publication June 19, 1998.
Revision received October 6, 1998.
Accepted for publication October 16, 1998.
 |
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