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J. Biol. Chem., Vol. 276, Issue 43, 40225-40233, October 26, 2001
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From the Dipartimento di Sanitá Pubblica e Biologia
Cellulare, Sezione di Anatomia, Universita' degli Studi di Roma Tor
Vergata, via O. Raimondo 8, 00173 Rome, Italy
Received for publication, June 5, 2001, and in revised form, July 13, 2001
In vitro addition of stem
cell factor (SCF) to c-kit-expressing A1-A4
spermatogonia from prepuberal mice stimulates their progression into
the mitotic cell cycle and significantly reduces apoptosis in these
cells. SCF addition results in a transient activation of extracellular
signal-regulated kinases (Erk)1/2 as well as of phosphatidylinositol
3-kinase (PI3K)-dependent Akt kinase. These events are
followed by a rapid re-distribution of cyclin D3, which becomes
predominantly nuclear, whereas its total cellular amount does not
change. Nuclear accumulation of cyclin D3 is coupled to transient
activation of the associated kinase activity, assayed using the
retinoblastoma protein (Rb) as a substrate. These events were followed
by a transient accumulation of cyclin E, stimulation of the associated
histone H1-kinase activity, a delayed accumulation of cyclin A2, and Rb
hyper-phosphorylation. All the events associated with SCF-induced cell
cycle progression are inhibited by the addition of either a PI3K
inhibitor or a mitogen-activated protein-kinase kinase (MEK) inhibitor,
indicating that both MEK and PI3K are essential for c-kit-mediated
proliferative response. On the contrary, the anti-apoptotic effect of
SCF is not influenced by the separate addition of either MEK or PI3K inhibitors. Thus, SCF effects on mitogenesis and survival in c-kit expressing spermatogonia rely on different signal transduction pathways.
The tyrosine kinase receptor encoded by the c-kit gene
and its ligand stem cell factor
(SCF)1 play a fundamental
role in gametogenesis (1). Most mutations of either c-kit or
SCF genes (W and Steel mutations, respectively) result in the loss of primordial germ cells in the embryonal gonad, whereas some Steel mutations affect gametogenesis after
birth (2-3).
c-kit expression is high in primordial germ cells and is down-regulated
in germ cells of the fetal gonad at around 13.5 days postcoitum (4). It
is resumed in perinatal oocytes at the end of meiotic prophase and in
proliferating spermatogonia at around 6 days postpartum (5-7). In the
adult testis, c-kit expression is absent in undifferentiated
spermatogonia (8), high in differentiating spermatogonia from type
A1 to B (5-7, 8), and turned off in meiotic and
postmeiotic cells (6-7). A truncated form of the c-kit kinase,
possibly playing a role during sperm-induced egg activation at
fertilization, is expressed during spermiogenesis (9-12).
c-kit expression in differentiating spermatogonia has led to the
hypothesis that the SCF/c-kit interaction is required for the
proliferation and/or survival of these cells. Several lines of evidence
support this hypothesis. In vivo injection of antibodies directed against the extracellular region of c-kit selectively blocks
proliferation and induces apoptosis of c-kit expressing type A
spermatogonia but not of c-kit negative undifferentiated spermatogonia
(7, 13). Furthermore, a mutation in the c-kit docking site for the p85
subunit of phosphatidylinositol 3-kinase (PI3K), introduced by a
knock-in strategy, causes a dramatic reduction of the spermatogonial
population in the prepuberal testis (14-15). A loss of spermatogonia
during postnatal development is also observed in a peculiar
Steel mutation, Sl17H (3). Finally,
in vitro addition of SCF, which is expressed by Sertoli
cells (16-17) under FSH control (17-18), selectively stimulates DNA
synthesis in type A but not in type B spermatogonia (17, 19).
The series of molecular events leading to G1 progression,
G1/S transition, and mitosis have been established in
several somatic cell types synchronized in G0 through serum
starvation (20-23). Synthesis of D-type cyclins and the assembly and
nuclear translocation of cyclin D/cyclin-dependent kinase
4/6 (cdk4/6) complexes is required for commitment to G1
entry, whereas the consequent cyclin E accumulation and activation of
the associated cyclin-dependent kinase 2 (cdk2) allows
progression through G1 (20-23). Cyclin D·cdk4/6 complexes trigger initial phosphorylation of the retinoblastoma protein
(Rb) and titrate cdk2 inhibitors (cip1/kip1 family),
thus de-repressing cyclin E/cdk2 activity. Hyperphosphorylation of Rb
by cyclin E/cdk2 is followed by release of the Rb-associated transcription factor E2F, which activates cyclin E transcription in a
positive feedback loop, allowing the burst of cyclin E accumulation and
activity in a narrow window coincident with the G1/S
transition. E2F transcriptional activity is required to elicit timely
induction of genes required for S phase progression, such as cyclin A2. Progression through the S phase coincident with the appearance of
cyclin A2/cdk2 activity is followed by rapid down-regulation of cyclin
E levels (20-23).
We report evidence that SCF acts as a mitogenic factor in cultured
c-kit-expressing spermatogonia and that both mitogen-activated protein
kinase kinase (MEK)- and PI3K-dependent pathways are
required for the proliferative response. The mitogenic effect is not
accompanied by an increase in total cellular amount of cyclin D3 (24),
but it is associated with a rapid change in its subcellular
localization. We also show that SCF is an anti-apoptotic factor for
spermatogonia, but the MEK- or the PI3K-dependent pathways
are not sufficient on their own to promote the survival response.
Isolation and Culture of Mouse Spermatogonia--
Spermatogonia
were obtained from either 5/6- or 8-day-old Swiss CD-1 mice, as
reported previously (17). Spermatogonial stem cells and proliferating
but undifferentiated spermatogonia are the prevalent germ cell types at
5-6 days of age, whereas differentiating (type
A1-A4, intermediate, and type B) spermatogonia
predominate at 8 days of age (25-26). Briefly, germ cell suspensions
were obtained by sequential collagenase-hyaluronidase-trypsin
digestions of freshly withdrawn testes from 20 animals. To release
cells completely, after the trypsin treatment, the pellet was
resuspended in 1 ml of culture medium and pipetted at least 30 times
and then brought to 20 ml with culture medium adding 2 mg/ml DNase and
10% fetal calf serum. Cell suspension was plated in Petri dishes (5 ml/dish) for 3 h in a humidified incubator at 32 °C to promote
adhesion of somatic cells. At the end of this pre-plating treatment,
enriched germ cell suspensions were washed from fetal calf serum, and
spermatogonia were then cultured in Eagle's minimal essential
medium supplemented with 1 mM DL-lactic acid, 2 mM sodium pyruvate, non-essential amino acids (Life
Technologies, Inc.). For time course experiments, spermatogonia were
either left untreated or stimulated with SCF (100 ng/ml, Genzyme) at
different time points and then they were processed as described below.
Where indicated cells were also incubated 1 h before SCF addition
with 10 µM U0126 (catalog number V1121, Promega),
with 10 µM LY294002 (catalog number 270-038-M005, Alexis), or with 1 µM tyrphostin AG490 (catalog number
658401, Calbiochem), all dissolved in Me2SO. In
these experiments, an equal volume of the Me2SO solvent was
also added in control and SCF-treated cultures. Nuclear morphology of
spermatogonia after the pre-plating time and after 24 h of culture
in the absence or constant presence of SCF and/or the signaling
inhibitors was assessed after hypotonic shock of 105 cells
(75 mM KCl) followed by fixation in methanol:acetic acid solution (3:1). Cells were then dropped onto glass slides to allow spreading of the nuclei and stained with Giemsa solution. Spermatogonia nuclei were judged as in interphase, metaphase, or apoptotic and counted from quadruplicate experiments. Somatic nuclei were excluded from the counts, and purity of spermatogonia was assessed as about 85%
after the pre-plating treatment and almost 100% after 24 h of culture.
DNA synthesis was studied by [3H]thymidine incorporation
followed by autoradiography as previously described (17). In these experiments, incubation with [3H]thymidine was performed
during the last 4 h of the 24 h culture period.
Western Blot Analysis and Antibodies--
Cells were harvested,
washed in cold PBS, and homogenized at 4 °C in lysis buffer
containing 10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 10 mM Immunoprecipitation and Cdks Kinase Assays--
2 × 106 viable cells were harvested and homogenized in 40 µl
of a modified lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10%
glycerol, 1 mM dithiothreitol, 0.1% Tween 20, 10 mM Immunofluorescence Analysis and TUNEL Assays--
Control and
1-h SCF-treated spermatogonia, preincubated or not with U0126 or
LY294002, were spotted onto poly-L-lysine glass slides and
fixed for 10 min at room temperature in 2% paraformaldehyde. Cells
were washed in PBS, permeabilized 10 min with PBS, 0.1% Triton X-100
and incubated for 30 min at room temperature with PBS, 0.5% bovine
serum albumin. Cells were incubated overnight at 4 °C in a
humidified chamber with mouse monoclonal anti-cyclin D3 antibody at a
final concentration of 2 µg/ml and then 1 h at room temperature
with cyanin 3-conjugated anti-mouse IgG (Calbiochem). Slides were
washed and mounted in 50% glycerol in PBS and immediately examined by
fluorescence microscopy. Nuclei were counterstained with 1 µg/ml
Hoechst (catalog number 33342, Sigma). Control experiments were
performed using mouse non-immune IgGs instead of the specific antibody.
For in situ detection of apoptotic cell death, control and
SCF-treated spermatogonia, preincubated or not with U0126, LY294002, or
AG490, after a 24-h period of culture were spotted onto
poly-L-lysine glass slides, fixed for 10 min at room
temperature in 2% paraformaldehyde, and subjected to TUNEL assay with
an in situ cell death detection kit (catalog number 1684817, Roche Molecular Biochemicals) by following the manufacturer's
instructions. Nuclei were counterstained with 1 µg/ml Hoechst
(catalog number 33342, Sigma). Slides where then examined by
fluorescence microscopy.
SCF Stimulates Cell Cycle Progression of c-kit Expressing
Spermatogonia through Both MEK and PI3K Signals--
Cultures of germ
cells obtained from 8-day-old mice are particularly enriched in
differentiating spermatogonia (25, 26), which express high levels of
c-kit (5-8, 14). Fig. 1 shows that,
after 24 h of culture, several nuclei with characteristic features
of apoptosis, such as reduced size and intense chromatin staining, can
be observed in untreated cells. In SCF-treated cultures, the frequency
of such cells is clearly reduced (see the last paragraph of this
section), and a clear increase in the number of mitotic figures (nuclei
showing condensed metaphase chromosomes) can be appreciated. These data
confirm that SCF is required to maintain the proliferative state of
differentiating spermatogonia cultured in vitro (17,
24).
We studied DNA synthesis and cell cycle progression in these cultures
by using [3H]thymidine incorporation and metaphase
counting. SCF induces a 2-fold increase in the number of
[3H]thymidine incorporating cells and a 3-fold increase
of metaphase counts with respect to the control after 24 h of
culture (Table I). We also analyzed the
effects of SCF addition in germ cell populations from 5- to 6-day-old
mice, when undifferentiated spermatogonia are the predominant cell
types (25, 26), and c-kit expression is not detectable (5, 7, 8, 27,
28). No stimulation of cell cycle progression was observed in these
cells (% of 3H-labeled cells in control cultures,
9.20 ± 0.05; in SCF-treated cultures, 9.40 ± 1.85; % cells
in mitosis in control cultures, 1.25 ± 0.35; in SCF-treated
cultures, 0.70 ± 0.30).
c-kit signaling pathways activated in cell cycle progression have been
shown to involve PI3K, MEK, and Janus-activated kinase 2 (JAK2) in
different cell types (14, 15, 29, 30). In mouse spermatogonia, PI3K
activation has been shown to be involved in SCF-dependent
proliferation (14, 15, 24); however, the possible involvement of MEK-
and JAK2-dependent pathways has not been studied. To
investigate whether these c-kit-activated signaling pathways mediate
the mitogenic activation of spermatogonia observed in vitro,
the proliferation assays were performed in the presence of inhibitors
selective for each of the three different pathways: the MEK inhibitor
U0126, the JAK2 inhibitor tyrphostin AG490, and the PI3K inhibitor
LY294002 (Table I). The inhibition of the MEK pathway abolished the
SCF-induced increase in both [3H]thymidine incorporation
and metaphase counts, demonstrating that the integrity of this pathway
is required for SCF induction of mitogenesis. Inhibition of PI3K
pathway also abolished SCF mitogenic effect, indicating that both MEK
and PI3K pathways are required. On the contrary, inhibition of JAK2
signaling had no effect on SCF-stimulated [3H]thymidine incorporation.
SCF Activates Both Extracellular Signal-regulated Kinases (Erk)1/2
and Akt Kinases in c-kit-expressing Spermatogonia--
Since the MEK
and PI3K inhibitors were effective in the inhibition of SCF-induced
proliferation of spermatogonia, we studied the Erk1/2 and PI3K
activation pathways induced by SCF in these cells in a time course
experiment. Fig. 2A shows that
MEK was activated as early as 5 min from the addition of SCF, since an increase of phospho-Erk1/2 could be detected with respect to the control. The activation of both Erks was maximal at 15 min and then
decreased to the control levels within 1 h, showing that SCF
induces a transient Erk1/2 activation. SCF-induced increase of
phospho-Erks is specifically regulated by MEK activation, since in the
presence of U0126 the phospho-Erk1/2 bands were no longer detectable
(Fig. 2B). To study the activation of PI3K, we monitored the
phosphorylation state of its substrate, the Akt kinase. SCF stimulation
induces a rapid and persistent Akt phosphorylation (Fig.
2C). Inhibition of PI3K with LY294002 completely blocked SCF-induced Akt phosphorylation (Fig. 2D). The two signaling
pathways were independently regulated by SCF, since the presence of
LY294002 or U0126 did not interfere with Erk1/2 or Akt activation,
respectively (Fig. 2, B and D). This result
indicates that, even though both MEK and PI3K activities are required
for SCF-induced mitogenic effect in spermatogonia, no cross-activation
occurs between these two signaling pathways in response to SCF
stimulation.
As expected from the observation that SCF did not induce an increase of
proliferation in germ cells from 5- to 6-day-old mice, SCF addition did
not modify the phosphorylation state of Erk1/2 in these cells at any
time point studied (Fig. 2E).
SCF Induces a Very Rapid G1/S Transition through
Sequential Induction of Cyclin E and Cyclin A2--
To study the
effect of SCF addition on the spermatogonial cell cycle, we analyzed
the expression of cyclins specifically expressed during the
G1/S phase by Western blot. SCF addition did not modify the
levels of cyclin D3, a D-type cyclin that is predominantly expressed in
proliferating spermatogonia (31, 32), at any time point studied (Fig.
3A), nor c-Myc levels (Fig.
3B), which are often up-regulated during mitogenic
stimulation in other cell types (20-23). However, the levels of cyclin
E were up-regulated after 1 h from SCF addition and decreased
after 3 h (Fig. 3C). Cyclin A2, which is expressed in
proliferating spermatogonia (33), was up-regulated between 10 and
16 h after SCF addition, and it returned to the control levels
after 24 h (Fig. 3D).
To exclude the possibility that in vivo exposure of
spermatogonia to endogenous SCF had already caused sustained expression of cyclin D3 prior to their isolation for the in vitro
culture, we stimulated spermatogonia with SCF following an overnight
incubation in the absence of the growth factor. Even under these
conditions, cyclin D3 did not increase upon SCF stimulation at any time
point checked (Fig. 3E). The levels of cyclin A2, however,
reached a maximum about 12 h after SCF addition (Fig.
3F).
SCF Treatment Causes a Transient Increase in the Activity of Cyclin
D3- and Cyclin E-associated Kinase Activities--
The increase of
cyclin E and subsequently of cyclin A2 levels in the absence of
detectable cyclin D3 quantitative modifications prompted us to
investigate whether the activity of cyclin D3·cdk4 complex was
affected by SCF addition. It is known that D-type cyclins induced by
growth factors activate cyclin-dependent kinases (cdk4 and
cdk6) to initiate Rb phosphorylation, which is then completed by cyclin
E/cdk2 and cyclin A/cdk2 (20-23). Western blot analysis of
spermatogonial extracts with a mouse cross-reactive monoclonal anti-Rb
antibody directed against aa 332-344 of human Rb showed that
hyperphosphorylation of Rb (the slower migrating bands) started to be
detectable as early as 2 h after SCF addition and reached a
plateau after 16 h (Fig.
4A). Similar results were obtained using a polyclonal anti-Rb antibody directed against a peptide
corresponding to 15 aa at the carboxyl terminus of human Rb (data not
shown). This indicates that the different mobility of Rb in SCF-treated
and control samples is actually due to changes in the phosphorylation
state of Rb and not to a change in its molecular size due to
proteolytic cleavage at the carboxyl terminus (a phenomenon which is
often associated to apoptotic death in some cell types). Rb was timely
hyperphosphorylated after SCF treatment also in spermatogonial cultures
that had been subjected to overnight growth factor deprivation (Fig.
4B).
In order to test whether the increase of Rb phosphorylation was due to
cyclin D3/cdk4 activation by SCF, cell extracts from spermatogonia were
immunoprecipitated with anti-cyclin D3 antibodies, and then a kinase
assay using GST-Rb as a substrate was performed. Fig.
5A shows a significant
increase of cyclin D3-associated Rb-kinase activity after 1 h of
stimulation with SCF, which became less evident after the 2nd h of
culture. As a control, we performed similar kinase assays on cyclin D3
immunoprecipitates using histone H1 as a substrate, and no stimulation
of H1 phosphorylation was observed after SCF addition (data not
shown).
U0126 or LY294002 abolished SCF-induced stimulation of cyclin
D3-associated Rb kinase activity (Fig. 5B), but they did not modify total cellular levels of cyclin D3 and cdk4 (Fig.
5C). Thus, both the MEK and the PI3K pathways converge at
the level of regulation of cyclin D3-dependent kinase
activity, rather than at the level of cyclin D3 or cdk4 synthesis or stabilization.
As expected from the time course of cyclin E accumulation in
SCF-treated cells (Fig. 3C), cyclin E/cdk2 kinase activity,
monitored using histone H1 as a specific substrate, was strongly
induced after 1 h in the presence of SCF and decreased to the
control levels after 4 h of stimulation (Fig. 5D).
SCF-induced cyclin E accumulation was also abolished after pretreatment
with either U0126 or LY294002 (Fig. 5E).
SCF Induces Nuclear Localization of Cyclin D3 through MEK- and
PI3K-dependent Pathways--
The subcellular localization
of cyclin D3 was studied by immunofluorescence experiments. As shown in
Fig. 6, the immunofluorescence staining
of spermatogonia using an anti-cyclin D3 antibody revealed that SCF
induced a marked increase in nuclear localization of the cyclin in the
majority of the cell population after 1 h, while in the control
cultures the fluorescence was restricted to the narrow ring of
cytoplasm typical of this cell population. Pretreatment with U0126 or
LY294002 completely abolished the nuclear accumulation of cyclin
D3-induced by SCF in the 1st h of culture (Fig. 6) suggesting that both
MEK and PI3K pathways are involved in promoting cyclin D3 nuclear
relocation upon activation of the SCF receptor. No change in the
subcellular localization of cyclin E, which is predominantly nuclear,
was observed after SCF addition (data not shown).
SCF-mediated Prevention of Spontaneous Apoptosis in Cultured
Spermatogonia Is Not Blocked by the Separate Inhibition of MEK- or
PI3K-dependent Pathways--
It has been proposed that SCF
acts as a survival factor that prevents apoptosis in differentiating
spermatogonia (13, 19, 34). In line with this we observed that during
the 24 h of culture, concomitantly with the increase of the
metaphase counts, SCF decreased the frequency of cells showing
morphologies typical of apoptosis (Fig. 1). Such effect was quantified
by TUNEL staining, which specifically detects DNA fragmentation
associated with apoptotic cell death (Fig.
7 and Table
II). In order to verify whether the mitogenic and antiapoptotic effects of SCF on spermatogonia share common activation pathways, we explored the possible involvement of
PI3K and/or MEK/Erk in SCF-mediated prevention of apoptosis. Contrary to what we observed about the mitogenic effect, neither the MEK- nor the PI3K-dependent pathway was essential for
the activation of the survival response, since preincubation with either U0126 or LY294002 of SCF-treated spermatogonial cultures does
not interfere with the antiapoptotic effect of SCF (Fig. 7 and Table
II). Similarly, addition of the JAK2 inhibitor tyrphostin AG490 has no
detectable effect. Only the simultaneous addition of MEK and PI3K
inhibitors partially reverts the SCF-activated survival response (Table
II).
In this paper we show that two signal transduction pathways are
involved in c-kit-induced proliferation of cultured spermatogonia. SCF
addition results in a transient activation of Erk1/2 kinases and in a
parallel activation of the PI3K-dependent Akt kinase. Inhibition of either the MEK or the PI3K signaling completely abolished
SCF-induced DNA synthesis and cell cycle progression, whereas
inhibition of JAK2-dependent pathways had no effect.
SCF stimulation of germ cell proliferation is not followed by phenomena
that are commonly observed in G0-G1-arrested
somatic cells after addition of different mitogenic stimuli (20-23).
SCF does not increase cyclin D3 or c-Myc cellular levels, and it does not affect levels of other positive or negative regulators of the
G1/S transition, such as the cdc25a phosphatase or the cdk inhibitors of both the Cip1/Kip1 and the Ink family (data not shown). However, SCF induces a marked change in the subcellular localization of cyclin D3; within the 1st hour of SCF treatment, cyclin
D3, which is predominantly cytoplasmic in control cells, becomes highly
concentrated in the nucleus. Nuclear translocation of D-type cyclins
has been also reported in the case of cAMP-dependent proliferation of primary thyrocytes (35) and
17 SCF-induced nuclear accumulation of cyclin D3 in spermatogonia is
coincident with a transient induction of its associated Rb kinase
activity. These events are followed by a very rapid induction of the
G1/S transition, monitored through transient accumulation
of cyclin E at very early times of culture and activation of its
associated histone H1 kinase activity, followed by induction of cyclin
A2 (a marker of the S phase) at later times, and progressive hyperphosphorylation of Rb.
The observation that SCF mitogenic stimulus provokes such a rapid
activation of the G1/S transition in differentiating
spermatogonia is in agreement with pioneering autoradiographic studies
by Monesi (37) on DNA synthesis in these cells, showing that duration of the "resting phase preceding DNA synthesis" (i.e. the
G1 phase) is very short, ranging between 2 and 3 h.
Inhibition of either MEK or PI3K signaling completely abolished
SCF-induced increase in nuclear localization of cyclin D3, cyclin
D3-associated Rb-kinase activation, cyclin E induction, and cell cycle
progression in c-kit-expressing spermatogonia. Thus, the
contemporaneous activation of both these pathways by SCF is essential
to trigger G1/S transition in these cells.
MEK and PI3K cooperation in promoting cell proliferation has been
explained by the observation that MEK-dependent Erk
stimulation often promotes the synthesis whereas
PI3K-dependent Akt activation leads to the stabilization of
D-type cyclins (20-23). Here we show a novel effect of the cooperation
between these two pathways, culminating in modulation of the
subcellular localization, rather than of total cellular levels, of a
D-type cyclin.
It has been shown that Erk activation can trigger a transient induction
of p21Cip1/Waf1 (38), which in turn can play a
positive role in the assembly, in the nuclear translocation, and in the
activation of cyclin D·cdk4/6 complexes (39). However, we found that
p21Cip1/Waf1 is barely detectable in spermatogonia
at early times of culture, and SCF treatment does not cause any
increase in its cellular levels (data not shown). Alternatively,
Erk-dependent pathways might regulate phosphorylation of
cyclin D3 residues homologous to Thr-156 of cyclin D1, whose mutation
is known to inhibit nuclear import of the cyclin D1·cdk4 complexes
(40).
The nuclear localization of D-type cyclins is also regulated by the
PI3K pathway through the inhibition of glycogen synthase kinase 3 It has been reported that mouse spermatogonia isolated from 5-day-old
mice and propagated on a feeder layer for an undefined period express
higher levels of cyclin D3 when stimulated with SCF, and this would
correlate with stimulation of DNA synthesis (24). In the present study
we demonstrate that primary cultures of spermatogonia freshly isolated
from 8-day-old mice are fully responsive to SCF, but no increase in
cyclin D3 levels can be observed, even when SCF treatment is performed
after overnight growth factor deprivation. Moreover, we found that
freshly isolated spermatogonia from 5- to 6-day-old mice are not
stimulated by the growth factor. The possibility exists that, even
though the cell population used by Feng et al. (24) should
not express c-kit at the beginning of culture (5, 7), it could
eventually acquire SCF responsiveness during the co-culture period.
Our data are in agreement with two recent reports (14, 15) in which
mutant mice were generated in which the c-kit codon tyrosine 719 (the
docking site for the p85 subunit of PI3K) was converted to
phenylalanine. The Y719F mutation induced a sex- and tissue-specific
defect in postnatal gametogenesis, since males are completely sterile
(14, 15). A complete block of DNA synthesis was observed in germ cells
at 8 days of age, when c-kit expressing differentiating type
A1-A4 spermatogonia are present and
predominant (14). Our in vitro results show that, in
addition to activating the PI3K pathway, SCF must also induce a
transient Erk activation in order to elicit proliferation of spermatogonia.
It has been proposed that SCF acts merely as a survival factor that
prevents apoptosis in differentiating spermatogonia, which are assumed
to be intrinsically committed to proliferate (13, 19, 34). We actually
found that SCF addition also partially inhibits apoptosis occurring in
germ cells from 8-day-old mice after 24 h of culture. However, the
anti-apoptotic effect observed in vitro was not inhibited by
the separate addition of either the MEK- or of the PI3K inhibitor,
whereas both inhibitors on their own can impair the mitogenic response.
Thus, the Erk1/2 activation and the PI3K-mediated Akt activation that
we observed in cultured spermatogonia are not essential for SCF
inhibition of apoptosis. Distinct SCF-activated signal transduction
pathways must be involved in the pro-survival response, since even the simultaneous addition of both MEK- and PI3K inhibitors does not completely suppress SCF anti-apoptotic effect. In agreement with our
in vitro observations, abolishment of c-kit-mediated PI3K signaling in c-kit Y719F knock-in mice was not associated to increased apoptosis in spermatogonia at 8 days of age (14).
In conclusion, our data indicate that soluble SCF stimulates
proliferation of c-kit expressing and differentiating type
A1-A4 spermatogonia in vitro
through both MEK- and PI3K-dependent pathways, by
triggering nuclear relocation of cyclin D3 and a rapid G1/S transition. Moreover, they show that the SCF-mediated proliferative and
survival effects on spermatogonia depend on the activation of different
combinations of intracellular signal transduction pathways.
We thank Drs. Massimo De Felici and Claudio
Sette for critical reading of the manuscript and useful suggestions.
*
This work was supported by grants from Ministero
dell'Universita' e della Ricerca Scientifica e Tecnologica and from
Agenzia Spaziale Italiana.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, August 13, 2001, DOI 10.1074/jbc.M105143200
The abbreviations used are:
SCF, stem cell
factor;
aa, amino acids;
Erk, extracellular signal-regulated kinase;
PI3K, phosphatidylinositol 3-kinase;
Rb, retinoblastoma protein;
MEK, mitogen-activated protein-kinase kinase;
PBS, phosphate-buffered
saline;
GST, glutathione S-transferase;
TUNEL, terminal dUTP
nick-end labeling;
cdk, cyclin-dependent kinase.
Signaling through Extracellular Signal-regulated
Kinase Is Required for Spermatogonial Proliferative Response to
Stem Cell Factor*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 0.1 mM sodium vanadate, and 1/100th (v/v) of a pre-formed
protease inhibitors mixture (P8340, Sigma). Total cellular proteins
were transferred to polyvinylidene difluoride membranes after SDS-PAGE. Membranes were blocked with PBS buffer containing 5% fat-free milk and
0.1% Tween 20 for 1 h at room temperature and then hybridized with primary antibodies. After hybridization with secondary antibodies conjugated to horseradish peroxidase, the immunocomplexes were detected
with Supersignal West Pico detection reagent (Pierce). Primary
antibodies used are as follows: anti-phospho-Akt and anti-Akt rabbit
polyclonal (PhosphoPlus Akt (Ser-473) antibody kit, catalog number
9270, New England Biolabs Inc.); anti-phospho Erk1/2 mouse monoclonal
antibody (sc-7383, Santa Cruz Biotechnology, Inc.); anti-Erk2 rabbit
polyclonal (sc-154, Santa Cruz Biotechnology, Inc.); anti-cyclin D3
mouse monoclonal antibody (sc-6283, Santa Cruz Biotechnology, Inc.);
anti-cyclin E rabbit polyclonal antibody (sc-481, Santa Cruz
Biotechnology, Inc.); anti-cyclin A2 rabbit polyclonal antibody
(sc-751, Santa Cruz Biotechnology, Inc.); anti-Rb (aa 332-344) mouse
monoclonal antibody (14001A, PharMingen); anti-Rb (carboxyl terminus)
rabbit polyclonal antibody (sc-50, Santa Cruz Biotechnology Inc.);
anti-p21Cip1/Waf1 mouse monoclonal antibody (catalog
number sc-6246, Santa Cruz Biotechnology, Inc.); anti-c-Myc rabbit
polyclonal antibody (sc-788, Santa Cruz Biotechnology, Inc.); anti-cdk4
goat polyclonal antibody (sc-260-G, Santa Cruz Biotechnology,
Inc.).
-glycerophosphate, 1 mM NaF, 0.1 mM
sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride)
containing 0.4 µl of a pre-formed protease inhibitors mixture (P8340,
Sigma). One hundred µg of proteins from clarified supernatants of
whole cell lysates were incubated with 20 µg/ml anti-cyclin D3
monoclonal antibody or anti-cyclin E polyclonal antibody for 2 h
at 4 °C on a rotating shaker. The immunocomplexes were recovered
with protein G-Sepharose or protein A-Sepharose (Sigma), respectively,
for 1 h at 4 °C, washed three times at 4 °C with PBS, 0.5%
bovine serum albumin and once with the specific kinase reaction buffer
(50 mM Hepes, pH 7.5, 10 mM MgCl2,
and 1 mM dithiothreitol). Kinase assays were performed at
30 °C for 30 min in a 20-µl volume of kinase reaction buffer
containing 10 mM -glycerophosphate, 0.1 mM
sodium vanadate, 0.2 µl of a pre-formed protease inhibitors mixture
(P8340, Sigma), 2.5 mM EGTA, 50 µM ATP, 0.1 mM protein kinase A inhibitor, 3 µCi of
[
-32P]ATP/reaction, and the following specific
substrates: 0.5 µg/reaction histone H1 (type III-S, Sigma) for cyclin
E/Cdk2 and 0.5 µg/reaction GST-Rb (sc-4112, Santa Cruz Biotechnology
Inc.) for cyclin D3/Cdk4/6. Reactions were terminated by addition of
4× Laemmli buffer. Samples were boiled, and proteins were separated by
SDS-PAGE. Phosphorylated substrates were visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Morphological aspects of
spermatogonial cell populations from 8-day-old mice stained with Giemsa
after 24 h of in vitro culture in the absence or
presence of SCF. Arrows indicate representative examples of
nuclei with characteristic features of apoptosis, such as reduced size
and intense chromatin staining. Asterisks indicate condensed
metaphase chromosomes typical of mitotic nuclei.
MEK- and PI3K-dependent stimulation of DNA synthesis and
cell cycle progression induced by SCF in spermatogonia from
8-day-old mice cultured for 24 h
View larger version (26K):
[in a new window]
Fig. 2.
MEK- and PI3K-dependent signal
transduction pathways are activated following SCF stimulation of c-kit
expressing spermatogonia cultured in vitro.
A, the time course of SCF-induced Erk1/2 activation in
spermatogonia from 8-day-old mice was monitored through Western blot
analysis using specific anti-phospho-Erk antibodies. Blots were
stripped and reprobed with anti-Erk2 antibodies for loading control.
This experiment was repeated three times with similar results.
B, Erk1/2 activation after 15 min of SCF treatment is
blocked by a selective MEK inhibitor but not by a selective PI3K
inhibitor, as shown by Western blot analysis using specific
anti-phospho-Erk antibodies in cells that had been preincubated with
either U0126 or LY294002. Blots were stripped and reprobed with
anti-Erk2 antibodies for loading control. C, the time course
of Akt activation in spermatogonia from 8-day-old mice was monitored
through Western blot analysis using specific anti-phospho-Akt
antibodies. Blots were stripped and reprobed with anti-Akt antibodies
for loading control. D, Akt activation after 15 min of SCF
treatment is blocked by a selective PI3K inhibitor but not by a
selective MEK inhibitor, as shown by Western blot analysis using
specific anti-phospho-Akt antibodies in cells that had been
preincubated with either U0126 or LY294002. Blots were stripped and
reprobed with anti-Akt antibodies for loading control. E,
SCF does not induce Erk1/2 activation in undifferentiated
spermatogonia, as shown by Western blot analysis of germ cells from 5- to 6-day-old mice using specific anti-phospho-Erk antibodies. Blots
were stripped and reprobed with anti-Erk2 antibodies for loading
control.
View larger version (33K):
[in a new window]
Fig. 3.
Time course of the effect of SCF addition on
cellular levels of markers of the G1-S transition in
cultured spermatogonia from 8-day-old mice. Representative Western
blot analysis using anti-cyclin D3 antibody (A and
E), anti-c-Myc antibody (B), anti-cyclin E
antibody (C), and anti-cyclin A2 antibody (D and
F) of equal amount of cell extracts from freshly obtained
spermatogonia (A-D) or from spermatogonia that had been
previously cultured overnight in the absence of growth factors
(E and F). Similar results were obtained in five
separate experiments.
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[in a new window]
Fig. 4.
Time course of the effect of SCF
addition on the phosphorylation state of Rb in cultured spermatogonia
from 8-day-old mice. A, representative Western blot analysis
of equal amount of cell extracts from spermatogonia using a monoclonal
anti-Rb antibody directed against aa 332-344 of human Rb. Similar
results were obtained in six separate experiments. B,
representative Western blot analysis of equal amount of cell extracts
from spermatogonia that had been cultured for the indicated times, and
after that they had been left overnight in the absence of growth
factors, using the same antibody as in A. Similar results
were obtained in three separate experiments.
View larger version (43K):
[in a new window]
Fig. 5.
A rapid and transient activation of cyclin
D3-associated Rb kinase activity and cyclin E-associated H1 kinase
activity is induced by SCF treatment. A, representative
kinase assays on equal amounts of cyclin D3 immunoprecipitates using
GST-Rb as a substrate (see "Experimental Procedures" for details).
Immunoprecipitates (IP) were also subjected to Western blot
analysis showing that equal amounts of cyclin D3 were present in all
samples (lower panel). This experiment was repeated four
times with similar results. B, kinase assays using GST-Rb as
a substrate on cyclin D3 immunoprecipitates from equal amounts of
extracts obtained from cells that had been preincubated with selective
MEK or PI3K inhibitors. C, Western blot analysis using
anti-cdk4 and anti-cyclin D3 antibodies of equal amounts of the same
cell extracts utilized in B. D, kinase assays using histone
H1 as a substrate on cyclin E immunoprecipitates from equal amounts of
cell extracts. E, Western blot analysis using anti-cyclin E
antibodies in cells that had been preincubated with selective MEK or
PI3K inhibitors.
View larger version (34K):
[in a new window]
Fig. 6.
A rapid change in the subcellular
localization of cyclin D3 is induced by SCF treatment, and it is
abolished by pretreatment with either MEK or PI3K inhibitors.
Representative immunofluorescence study using specific anti-cyclin D3
antibodies. Hoechst counter-staining was used to control the
subcellular localization of cyclin D3. This experiment was repeated
five times with identical results.
View larger version (17K):
[in a new window]
Fig. 7.
SCF prevents apoptotic DNA fragmentation
induced by the cell culture condition in spermatogonia, and the
separate inhibition of either MEK- or PI3K-dependent
pathways has no effect on the survival response. Representative
TUNEL staining of spermatogonial cells cultured for 24 h (see
"Experimental Procedures" for details).
SCF-triggered inhibition of spontaneous apoptosis in spermatogonia from
8-day-old mice cultured for 24 h is partially impaired by the
simultaneous but not by the separate addition of MEK and PI3K
inhibitors
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol-dependent proliferation of the uterine
epithelium (36). Our data show that nuclear translocation of a D-type
cyclin can be stimulated also by the activation of a tyrosine kinase receptor.
exerted by Akt. Indeed, inhibition of glycogen synthase kinase
3-dependent phosphorylation of cyclin D1 at the Thr-286 residue is coupled to the maintenance of nuclear localization of this
cyclin during the G1/S transition (41-42). We suggest that a similar mechanism may regulate cyclin D3 localization in response to
SCF.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 39-06-72596272;
Fax: 39-06-72596268; E-mail:
pellegrino.rossi@med.uniroma2.it.
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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