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246
Research Article
Cdc2-mediated Schwann cell migration during
peripheral nerve regeneration
In Sun Han1,*, Tae Beom Seo1,*, Kwan-Hoi Kim2, Jin-Hwan Yoon3, Sung-Jin Yoon4 and Uk Namgung1,‡
1
Department of Oriental Medicine, Daejeon University, Daejeon 300-716, Korea,
Department of Pharmacology, School of Medicine, Pusan National University, Busan, Korea
3
Department of Sports and Leisure Studies, Hannam University, Daejeon 300-791, Korea
4
Department of Physical Education, Korea University, Seoul, Korea
2
*These authors contributed equally to this work
‡
Author for correspondence (e-mail: [email protected])
Accepted 30 October 2006
Journal of Cell Science 120, 246-255 Published by The Company of Biologists 2007
doi:10.1242/jcs.03322
Journal of Cell Science
Summary
Schwann cell migration facilitates peripheral nerve
regeneration after injury. We have recently found increased
activation of Cdc2 kinase in regenerating sciatic nerves.
Here we show that Cdc2 phosphorylation of caldesmon
regulates Schwann cell migration and nerve regeneration.
A robust but transient increase in Cdc2 expression was
found in cultured Schwann cells prepared from the sciatic
nerve in rats that had undergone crush injury for 7 days.
These ‘injury-preconditioned’ Schwann cells exhibited
enhanced migration compared with non-preconditioned
control cells and treatment with the cdk inhibitor
roscovitine prevented cell migration. After transduction
with recombinant Cdc2 DNA adenoviral vectors, Schwann
cells were implanted into sciatic nerves; those expressing
wild-type Cdc2 migrated further in the distal direction
than those expressing dominant-negative Cdc2. We
identified caldesmon as a downstream substrate of Cdc2 in
Schwann cells and its phosphorylation by Cdc2 changed its
subcellular localization. Overexpression of dominantnegative caldesmon significantly counteracted the
migration effect caused by Cdc2. Finally, neurite outgrowth
of cultured DRG sensory neurons, facilitated by co-culture
with injury-preconditioned Schwann cells, was suppressed
by roscovitine treatment. The results indicate that
activation of the Cdc2-caldesmon pathway is necessary for
Schwann cell migration and suggest a role for this pathway
in peripheral axonal growth.
Introduction
Peripheral nerve fibers after injury, unlike those in the central
nervous system, regrow towards their original target and can
recover functionally. This ability is attributed to intrinsic
neuronal properties and surrounding non-neuronal activities in
which Schwann cells play a major supportive role. Schwann
cells in the injured nerve area proliferate and migrate into the
distal end forming the band of Büngner and support axonal
regrowth (Fawcett and Keynes, 1990). Schwann cell migration,
which also occurs at proximal end of the injury area, provides
a guide for regenerating axons by interacting with nerve fibers
or basal lamina (Williams et al., 1983; Daniloff, 1991; Guenard
et al., 1992; Torigoe et al., 1996). Several studies showed that
Schwann cell migration is crucial for successful axonal
elongation (Torigoe et al., 1996; Anton et al., 1994a). Of the
molecular factors responsible for Schwann cell migration,
NGF and its Schwann cell receptor (low-affinity NGF receptor
or LNGFR) and 4C5 Schwann cell surface antigen were
reported to mediate Schwann cell migration on denervated
sciatic nerve (Anton et al., 1994a; Yfanti et al., 2004). Oxidized
galectin-1, which is produced from Schwann cells and neurons
and functions as a cytokine, and extracellular matrix
components, such as laminin (merosin) and products of
chondroitin 6-sulphotransferase activity, were reported to
promote axonal regeneration by Schwann cell migration
(Fukaya et al., 2003; Anton et al., 1994b; Liu et al., 2006).
These studies implicate the importance of the molecular
interaction of Schwann cells with the environment, but the
events within the migrating Schwann cell in response to injury
are largely unknown.
Cdc2 (or cdk1) is a prototypical cyclin-dependent kinase that
promotes G2-M phase transition in the cell cycle. The range of
Cdc2 kinase substrates is broad, implying its importance for
diverse cell functions including DNA replication and mitosis,
dynamic regulation of spindle assembly and actin polarization
(Ubersax et al., 2003). Moreover, Cdc2 in association with
cyclin B2 mediates cell migration in prostate cancer cells
(Manes et al., 2003). A function of Cdc2 and other cdk family
proteins in the nervous system has begun to emerge recently.
There have been extensive studies on the role of Cdk5 in neural
migration during CNS development, axon guidance, neuronal
degeneration and axonal regeneration (Dhavan and Tsai, 2001;
Patrick et al., 1999; Namgung et al., 2004). Stimulation and
activation of cdk family proteins including Cdc2 and Cdk2 in
postmitotic neurons are related to neuronal apoptosis or
degeneration (Rideout et al., 2003; Konishi and Bonni, 2003;
Vincent et al., 1997). By contrast, induction of Cdk2
expression and activation in non-neuronal cells in the nervous
system such as astrocytes, oligodendrocytes and Schwann cells
was shown to be associated with proliferation (Tikoo et al.,
2000; Tanaka et al., 1998; Belachew et al., 2002).
Given that Cdc2 activation mediates cell proliferation and
Key words: Migration, Schwann cells, Cdc2, Caldesmon, Axonal
regeneration, Sciatic nerve
Cdc2-mediated Schwann cell migration
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Journal of Cell Science
migration in non-neuronal systems, the functional
involvement of Cdc2 in the regenerating peripheral nerves
in which Schwann cells proliferate and migrate would be
expected. We have recently found that Cdc2 protein
expression and its activity were upregulated in sciatic
nerves undergoing axonal regeneration promoted by
physical training (Seo et al., 2006). Here we explored
Cdc2 function in Schwann cells associated with nerve
regeneration, and found that Cdc2 expression was induced
in Schwann cells and promoted Schwann cell migration,
which is functionally related to the nerve regeneration
process. We also present evidence of Cdc2-induced
caldesmon activation during Schwann cell migration.
Results
To investigate changes in Cdc2 expression, nerve segments
proximal and distal to the crush site were prepared. Cdc2
protein was not detected in intact nerves, yet its expression
increased strongly at 3 and 7 days post crush (d.p.c.)
followed by a decrease at 14 d.p.c. (Fig. 1A). Longitudinal
distribution of Cdc2 protein showed higher levels in the
distal portion of the nerve at 7 d.p.c. compared with that
at 3 d.p.c. (Fig. 1B). Similarly, Cdc2 mRNA was
transiently elevated at 3 and 7 d.p.c. showing an increase
in the distal segment at day 7 (Fig. 1C). Cdc2 mRNA
expression in the nerve was examined further by FISH
using a Cdc2 riboprobe. Cdc2 mRNA was clearly detected
in the sciatic nerve prepared at 7 d.p.c. and mostly
overlapped with S100␤ protein signals, which label
Schwann cells (Fig. 1D). FISH in either intact nerve tissue
or using a sense probe did not show any signal.
Examination of Cdc2 protein in the injured nerve by
immunofluorescence staining also revealed colocalization
with S100␤ (Fig. 1E). These data suggest the transient
induction of Cdc2 expression in Schwann cells of the
injured sciatic nerve.
To investigate Cdc2 function in Schwann cells, we first
examined whether Cdc2 expression was induced in
cultured Schwann cells prepared from injured sciatic
nerves. Cdc2 protein levels in the cultured Schwann cells,
which were prepared from the sciatic nerve in rats that had
undergone crush injury for 7 or 14 days (‘injurypreconditioned’ cells), were higher than those in Schwann
cells from intact nerve (‘non-preconditioned’ cells) (Fig.
2A). In injury-preconditioned cells, increases in Cdc2
protein were further upregulated by treatment of serum
and forskolin for cell proliferation. A portion of S100␤positive Schwann cell counts in the total cell population
was increased to ~90% by injury preconditioning or by
inducing proliferation, as demonstrated previously
(Morrissey et al., 1991). Double immunofluorescence
staining analysis further showed that the number of Cdc2positive cells in the total cell population or Schwann cell
population was elevated by nerve injury and/or by serum
and forskolin treatment (Fig. 2B), suggesting that
Schwann cells are the major cell type inducing Cdc2
expression in response to nerve injury.
Schwann cells in the injured peripheral nerves can
migrate into the injury site and promote axonal
regeneration. Here, we examined Schwann cell migration
in both explant culture of sciatic nerve segments and filter
Fig. 1. Induction of Cdc2 protein in regenerating sciatic nerves. (A) Western
analysis showed a strong but transient increase of Cdc2 protein in 2 cm
nerve segments proximal and distal to the injury site. (B) Distribution of
Cdc2 protein around the injury site. Vertical arrow indicates the injury site.
(C) RT-PCR for Cdc2 mRNA expression in 1 cm nerve segment proximal
(P) or distal (D) to the injury site. (D) FISH analysis of Cdc2 mRNA in the
sciatic nerve. Sciatic nerve sections were used for FISH with antisense or
sense riboprobe and immunofluorescence staining with anti-S100␤
antibody. (E) Immunofluorescence views show that signals for S100␤ and
Cdc2 proteins are colocalized in the nerve sections. In D and E, transverse
nerve sections (20 ␮m) 0.5 cm distal to the injury site were prepared at 7
d.p.c. Actin was detected in A-C as internal loading controls. Bars, 50 ␮m.
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Fig. 2. Cdc2 expression in cultured Schwann cells. (A) Western blot
analysis of Cdc2 in cultured Schwann cells. Schwann cells were
prepared from sciatic nerves given crush injury for 0-14 days or from
the sciatic nerve of the rat of postnatal day 3 (pnd3). Cells were
incubated for 2 days in 10% serum and 2 ␮M forskolin (FSK) (+) or
0.5% serum and 0 ␮M forskolin (–). Cdc2 protein in Schwann cells
was increased by injury preconditioning of the sciatic nerve and
further upregulated by serum and forskolin treatment. Actin was
detected as an internal loading control. (B) Merged views for the
localization of S100␤- or Cdc2-positive cells in the total cell
population (Hoechst 33258 stained) or Cdc2-positive cells in S100␤positive cell population. Schwann cells were prepared from sciatic
nerves with or without injury preconditioning for 7 days. Cells
positive to S100␤ and/or Cdc2 were increased by injury
preconditioning, and forskolin and serum treatment. Bar, 20 ␮m.
culture of cells from sciatic nerve trypsinates. Schwann cell
migration was strongly induced in the explant culture of injurypreconditioned sciatic nerve (Fig. 3A) whereas no visible cell
motility was observed in the control nerve. Analysis using
microfilter chamber culture showed that the number of migrated
Schwann cells was much higher in the injury-preconditioned
group than non-preconditioned control group (Fig. 3B).
Treatment with the cdk inhibitor roscovitine decreased
Schwann cell migration in a dose-dependent manner (Fig. 3C),
suggesting that cell migration depends on Cdc2 activity.
We further examined the role of Cdc2 activity in Schwann
cell migration in vivo. After infection with adenovirus
expressing wt- or dn-Cdc2 cDNA together with green
fluorescence protein (GFP) reporter, Schwann cells were
implanted into the injury site of the sciatic nerve. Infected
Schwann cells were identified in the sciatic nerve sections by
visualizing GFP. GFP-expressing Schwann cells were found at
different distances along the distal area of the injured sciatic
nerve, and mostly localized close to the nerve fibers (Fig. 4A).
At 3 d.p.c. and 7 d.p.c. cells expressing wt-Cdc2 migrated
further towards the distal end of the nerve compared with cells
expressing dn-Cdc2 (Fig. 4B,C).
Caldesmon is an actin-binding protein that is regulated by
phosphorylation by Cdc2 kinase (Yamashiro et al., 1991). Here
we investigated whether caldesmon participated in Schwann
cell migration in association with Cdc2 activation. Caldesmon
phosphorylation was increased in the injury-preconditioned
Schwann cells (Fig. 5A). Western blot analysis of phosphocaldesmon showed a positive band from Schwann cell lysate,
which had been immunoprecipitated with anti-actin antibody
and phosphorylated in vitro by Cdc2 kinase (Fig. 5B, lane 1),
but the band intensity was decreased by addition of roscovitine
or by depletion of Cdc2 enzyme in the kinase reaction (Fig.
5B, lanes 2 and 3). Caldesmon immunoprecipitated by actin
was further used for in vitro Cdc2 kinase assay. As shown in
Fig. 5C, phosphorylated caldesmon was detected, but the
addition of cdk inhibitor roscovitine or histone H1 as a
competitive substrate to the reaction decreased caldesmon
phosphorylation by Cdc2, indicating the reaction specificity of
Cdc2 phosphorylation of caldesmon. In injury-preconditioned
Schwann cells, cell counts for phospho-caldesmon-positive
immunostaining were significantly decreased in the cell
population transfected with dn-Cdc2 cDNA compared with
empty vector transfection (Fig. 5D). By western blot analysis,
we confirmed the downregulation of phospho-caldesmon levels
in Schwann cells transfected with dn-Cdc2. We then
investigated whether Cdc2 activity affected caldesmon
interaction with cytoskeletal proteins present in the periphery
of the cell. In injury-preconditioned Schwann cells, caldesmon
was concentrated in the central area of the cell. The treatment
of roscovitine changed the localization of caldesmon signal to
the periphery (Fig. 5E), suggesting that caldesmon interacts
with the peripheral cytoskeleton and is released by Cdc2
phosphorylation.
To determine the effects of Cdc2-mediated caldesmon
phosphorylation on Schwann cell migration, cells were
transfected with mutant forms of Cdc2 and caldesmon. A
plasmid construct expressing mutant caldesmon in which all
seven phosphorylation sites are missing has been developed
(Yamashiro et al., 2001) and was used here to inhibit the Cdc2mediated caldesmon pathway. In injury-preconditioned
Schwann cells, which were expected to express Cdc2, the
number of migrated cells was significantly lower when
transfected with either dn-Cdc2 or caldesmon 7th mutant than
vector control (Fig. 6A). In non-preconditioned Schwann cells,
migration was significantly facilitated by transfection with wtCdc2 DNA compared with control vector (Fig. 6B). However,
cell migration was decreased by cotransfection with Cdc2 and
caldesmon 7th mutant DNA, suggesting interference of
endogenous caldesmon phosphorylation by transfected mutant
caldesmon. Expression of wild-type caldesmon alone did not
increase cell migration, suggesting the possible requirement for
its activation by Cdc2.
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Cdc2-mediated Schwann cell migration
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Fig. 3. Cdc2 activity is required for Schwann cell
migration. (A) Immunofluorescence staining of
explant culture for the detection of S100␤-positive
Schwann cells (red) and Cdc2-positive cells (green).
Enlarged image of the rectangle is shown in the
inset. The merged image indicates that most
migrating Schwann cells in injury-preconditioned
explants express Cdc2. Quantification data of
Schwann cell migration in the explant culture with
or without injury preconditioning (Injury-pre. vs.
Non-pre) are shown on the right. The arrow
indicates the direction of migrating Schwann cells
from the sciatic nerve center. (B) Comparison of
Schwann cell migration in culture between injurypreconditioned and non-preconditioned groups. The
data represent Schwann cell migration as percentage
of total cells on the filter culture recovered from
injury-preconditioned or non-preconditioned sciatic
nerves. (C) Dose-dependent decrease of Schwann
cell migration by roscovitine treatment. Injurypreconditioned Schwann cells were cultured under
the different concentrations of roscovitine for 2
days. Statistical comparisons were made compared
with the cell group treated with 0 ␮M roscovitine
(*P<0.05; ***P<0.001). In B,C, migrated Schwann
cells on the coverslip were immunostained with
anti-S100␤ antibody, and the cells on the top of the
filter were stained with Cresyl Violet. The cells from
ten random fields were counted. (n=3 for A and n=4
for B,C). Bars, 50 ␮m (A); 20 ␮m (inset in A).
Cdc2 upregulation in migrating Schwann cells suggests
functional association with axonal regeneration. To delineate
the role of Cdc2 in axonal regeneration after injury, neurite
outgrowth of DRG sensory neurons in the presence of Schwann
cells was investigated. Sciatic nerves with injury
preconditioning facilitated neurite outgrowth of DRG sensory
neurons as demonstrated previously (Fig. 7A,B) (Smith and
Skene, 1997). We found that cultured sensory neurons prepared
from DRG without injury preconditioning showed extensive
neurite outgrowth when co-cultured with injurypreconditioned Schwann cells. However, incubation with
roscovitine significantly inhibited the development of neurite
branch points. Roscovitine also caused a 56% decrease in
neurite length but this was not statistically significant (P=0.57,
n=4; Fig. 7B).
Discussion
Our data provide new evidence that Cdc2 mediates Schwann
cell migration in the injured peripheral nerve and facilitates
axonal regeneration. We demonstrate in vitro and in vivo that
migration of injury-preconditioned Schwann cells was
significantly delayed by Cdc2 inhibition, and that they regained
motility by Cdc2 overexpression. Our data further showed that
Cdc2 phosphorylated caldesmon in Schwann cells, and
blockade of the Cdc2-caldesmon pathway suppressed cell
migration. Neurite outgrowth of cultured DRG sensory
neurons was largely improved by co-culture with injurypreconditioned Schwann cells.
We have recently reported increased activity of Cdc2 kinase
during axonal regeneration of the injured sciatic nerve
facilitated by treadmill training in the rat (Seo et al., 2006).
Here, we sought the physiological role of Cdc2 in Schwann
cells, which had been preconditioned to activate the
regeneration process. Cdc2 synthesis was increased in
Schwann cells that were prepared from sciatic nerves that had
undergone crush injury for 7–14 days. The change in Cdc2
protein levels showed a similar time course to that seen in the
injured sciatic nerve. Measurement of Schwann cell migration
in the explant culture and microfilter chamber showed strong
induction by injury preconditioning of the sciatic nerve.
Inhibition of Cdc2 activity by roscovitine or by expression of
dn-Cdc2 mutant resulted in decreased cell migration,
indicating the requirement of Cdc2 activity for cell migration.
A role of Cdc2 function in migration was further demonstrated
in Schwann cells that were infected with Cdc2-expressing viral
vectors and implanted into the sciatic nerve. Since transplanted
Schwann cells expressing wt-Cdc2 migrated faster toward the
distal end than those expressing dn-Cdc2, we presume that
endogenous Schwann cells in which Cdc2 expression was
increased by nerve crush might conduct similar motility along
the Wallerian degenerated trunk (Fukaya et al., 2003; Torigoe
et al., 1996).
Caldesmon is present as different isoforms in actin- and
calmodulin-binding proteins of smooth muscle and non-muscle
cells. Its binding to actin can be regulated by phosphorylation
by many kinases such as Erk, p38, Cdc2, and calmodulindependent protein kinase II (Childs et al., 1992; Ikebe and
Reardon, 1990; Yamashiro et al., 1991; Mirazapoiazova et al.,
2005). Non-muscle 83 kDa caldesmon is released from actin
filaments when phosphorylated by Cdc2 kinase (Yamashiro et
al., 1991). Increased levels of ␣v␤3 integrin in prostate cancer
cells upregulate Cdc2 phosphorylation of caldesmon in events
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Journal of Cell Science 120 (2)
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Fig. 4. Facilitated migration of
implanted Schwann cells infected
with Ad-Cdc2 viral vectors in the
injured sciatic nerve.
(A) Representative images of
Schwann cells infected with Ad-wtCdc2 and Ad-GFP in the sciatic
nerve sections. Seven days after
injury, infected Schwann cells
(marked in circles) were identified
by co-infected GFP reporter above
the background staining with NF200 or with Hoechst 33258 nuclear
staining. (B) Distribution of
implanted Schwann cells along the
nerve. In 3 d.p.c. and 7 d.p.c. groups,
Schwann cells infected with wtCdc2 virus were observed in the
more distal portion of the nerve
compared with those infected with
dn-Cdc2 virus. Arrow indicates site
of injury. (C) Quantitative
comparison of infected cells in the
distal portion of the sciatic nerves. In
the sciatic nerves prepared at 3 d.p.c.
or 7 d.p.c., statistical comparisons
were made between the groups
infected with wt-Cdc2 and dn-Cdc2
viruses (asterisks marked to the left
of the symbols indicating wt-Cdc2
viral infection; *P<0.05, **P<0.01,
*P<0.001; n=4, mean ± s.e.m.).
Bars, 100 ␮m (A); 300 ␮m (B).
that lead to increased cell migration (Manes et al., 2003). Since
cell proliferation and migration are the key events for
carcinogenesis, the study by Manes et al. raises an interesting
issue that a single molecule controlling the cell cycle can also
regulate cell migration (Juliano, 2003). Since Schwann cells
undergo the stages of proliferation and migration in the injured
peripheral nerve before differentiation stage for myelination,
the cell-cycle protein Cdc2 could be functionally linked to
migration. In Schwann cells, we found caldesmon
phosphorylation by Cdc2 and changes in its subcellular
localization by roscovitine treatment. Previous studies show
that caldesmon bound to actin filaments at the cell periphery
is released by its phosphorylation, and thus modulates cell
migration (Ishikawa et al., 1998; Manes et al., 2003;
Mirzapoiazova et al., 2005). In our study, addition of
roscovitine to injury-preconditioned Schwann cells changed
the subcellular localization of caldesmon protein to the
periphery, suggesting a dynamic regulation of its interaction
with actin via Cdc2. We further found that the blockade of
Cdc2-mediated caldesmon pathway by expression of
caldesmon 7th mutant cDNA significantly reduced cell
migration. Caldesmon 7th mutant was constructed by
substituting all Cdc2 phosphorylation site and its effects have
been well demonstrated in the regulation of actin and
calmodulin, and mitosis (Yamashiro et al., 1991; Yamashiro et
al., 1995; Yamashiro et al., 2001). Thus, Cdc2 phosphorylation
of caldesmon in Schwann cells appears to regulate cell motility
via modulation of the interaction with actin.
Our data suggest that Cdc2-mediated Schwann cell
migration may be involved in axonal elongation. DRG sensory
neurons when preconditioned by sciatic nerve injury showed
enhanced neurite outgrowth compared with nonpreconditioned control (Smith and Skene, 1997). In the present
study, non-preconditioned DRG sensory neurons displayed
significantly increased neurite extension by co-culturing with
injury-preconditioned Schwann cells, which was then
attenuated by roscovitine treatment. Facilitated Schwann cell
migration via the activation of Cdc2 signaling pathway might
contribute to guide neurite extension in culture as demonstrated
in regenerating axons in vivo (Torigoe et al., 1996). Although
the present data implicate a potential role of Cdc2 in axonal
elongation via Schwann cell migration, the importance of
Cdc2-induced Schwann cell proliferation deserves to be
mentioned. Cdk2 activity, which is necessary for the transition
from G1 to S phase, is important for Schwann cell proliferation
(Tikoo et al., 2000), and increased Cdk2 protein levels were
Cdc2-mediated Schwann cell migration
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Journal of Cell Science
Fig. 5. Cdc2 phosphorylation of caldesmon in injury-preconditioned
Schwann cells. (A) Western blot analysis of phospho-caldesmon in
cultured Schwann cells. Phospho-caldesmon protein was greatly
increased in cultured Schwann cells with injury preconditioning for 7
days. As a control, Schwann cell lysate was prepared from the sciatic
nerve in rats at postnatal day 3 (pnd3). (B) Identification of phosphocaldesmon protein in Schwann cells by western blot analysis.
Caldesmon pulled down by immunoprecipitation of actin was used as
substrate for the kinase reaction by exogenous Cdc2 enzyme (lane 1).
Kinase reaction in the presence of roscovitine is shown in lane 2 and
the reaction without Cdc2 in lane 3. (C) In vitro kinase assay for
caldesmon phosphorylation by exogenous Cdc2. Caldesmon pulled
down by immunoprecipitation of actin was used as a substrate for the
kinase reaction. Inclusion or exclusion of roscovitine (rosco) or histone
H1 in the incubation mixture are indicated respectively by + or –,
respectively. Caldesmon phosphorylation by Cdc2 was decreased by
the cdk inhibitor, roscovitine or by
competition with the alternative
substrate, histone H1. (D) Inhibition
of caldesmon phosphorylation by dnCdc2 expression. Schwann cells were
cotransfected with pGFP and pCMV5
vector or pGFP and pCMVdn-Cdc2.
A cell group transfected with dnCdc2 was less positive for phosphocaldesmon immunostaining than the
vector control. The graph shows a
significant reduction of phosphocaldesmon-positive (+) cells among
the GFP-positive (+) transfected cell
population by dn-Cdc2-transfection.
(n=3, mean ± s.e.m.). Western blot
analysis shows significant
suppression of phospho-caldesmon
immunopositivity in Schwann cells
that had been injury preconditioned
and transfected with dn-Cdc2.
(E) Subcellular localization of
caldesmon in injury-preconditioned
Schwann cells. The perinuclear
distribution of caldesmon in vehicletreated cultures contrasts with the
peripheral distribution in cultures
treated with 10 ␮M roscovitine
(arrow). Bars, 30 ␮m (D); 20 ␮m (E).
Actin was detected as an internal
loading control.
observed in the injured sciatic nerves (T.B.S. and U.N.,
unpublished observation). While these data suggest the
possible involvement of Cdk2 in peripheral nerve regeneration,
Cdc2 activity could be required for the complete cell-cycle
progression in Schwann cells. Indeed, our data indicate that
Cdc2 activity is positively associated with Schwann cell
proliferation (see Fig. 2). One possible mechanism explaining
the dual roles of Cdc2 for cell migration and cell-cycle
progression would be Cdc2 displacement and differential
activation by cyclin B1 and B2 between cytoplasm for
caldesmon phosphorylation and nucleus for M-phase
transition. In human cultured cells, cyclin B2 is found in the
Golgi complex and cell membrane, and unlike cyclin B1, is not
located in the nucleus at prophase (Jackman et al., 1995).
Whether cyclin B1 and cyclin B2 are similarly distributed in
Schwann cells and activate Cdc2 kinase to regulate cell
proliferation and migration at different stages of the cell cycle
remains to be determined.
In summary, we found that upregulation of Cdc2 protein in
Schwann cells is important for cell migration and has a growthpromoting activity for regenerating axons. Together with the
present finding, recent studies on Cdc2-mediated neuronal
apoptosis demonstrate a novel function of Cdc2 in the nervous
system (Konishi and Bonni, 2003; Konishi et al., 2002).
Moreover, we found upregulation of Cdc2 activity in a nonneuronal cell population of the injured spinal cord, which
positively correlated with the extent of axonal sprouting of
injured neurons (T.B.S. and U.N., unpublished observation),
which implicates the pathophysiological significance of Cdc2
in CNS function. Growing bodies of evidence show that nonneuronal cells such as astrocytes in the CNS could actively
participate in the recovery of injured spinal cord via enhanced
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Journal of Cell Science 120 (2)
serum] and 2 mM glutamine and 1% penicillin-streptomycin. Cells were incubated
for 48 hours before the harvest for immunofluorescence staining or western blot
analysis. In experiments for roscovitine inhibition of Cdc2 activity, injurypreconditioned Schwann cells were cultured in the presence of 5 or 10 ␮M
roscovitine or the equivalent volume of DMSO vehicle for 6 hours before harvest.
For DRG sensory neuron and Schwann cell co-culture, DRG sensory neurons
(1.5⫻102 cells/well) were cultured for 24 hours and Schwann cells (1⫻104/well)
were added. The co-cultures were maintained in 500 ␮l DMEM supplemented with
10% serum in the presence of 10 ␮M roscovitine or equivalent volume of 100% of
DMSO for 2 days before cell harvest. After immunofluorescence staining with anti␤III-tubulin antibody (1:200, TUJ1, rabbit polyclonal, Covance, Berkeley, CA),
digital images of neuronal process were captured and transferred to Adobe
Photoshop (version 5.5). The number and length of neurite processes exhibiting
clear outgrowth (longer than cell body size) from the cell body were analyzed by
using i-Solution software (Image and Microscope Technology, Goleta, CA). Mean
branch points and neurite length were determined by analyzing at least 50 sensory
neurons that were randomly selected in each experiment.
Journal of Cell Science
Cell migration assays
After preliminary culture for 3 days, Schwann cells (1⫻104) were seeded on Costar
filter chambers (bottom diameter: 6.5 mm, 8 ␮m pore size, Costar, Cambridge, MA)
which had been coated with poly-L-ornithine and laminin mixture. Cells were
allowed to grow on the surface of the chamber and to migrate to a precoated
coverslip (12 mm in diameter) beneath the chamber for 2 days. Migrated cells were
fixed with 4% paraformaldehyde and 4% sucrose in PBS for immunofluorescence
staining with anti-S100␤ antibody (1:200, Dako, Denmark) or Cresyl Violet staining
(0.2% cresyl violet in water for 25 minutes). Digital images of stained cells were
measured and transferred to Photoshop. The number of cells in each image was
counted by using i-Solution software program. A similar procedure was also applied
for digital image collection and quantitative analysis from the explant culture (see
below).
Explant culture
Fig. 6. Schwann cell migration is facilitated by activation of the
Cdc2-caldesmon pathway. Cultures of Schwann cells from nerves
with (A) or without (B) injury preconditioning were transfected with
plasmids expressing dn-Cdc2, wt-Cdc2, caldesmon 7th mutant (Cald
7th), and pGFP. All GFP-positive cells that migrated to coverslips
were counted. Horizontal lines in A and B indicate the mean number
of cells (**P<0.01, ***P<0.001; n=5).
migration (Okada et al., 2006) and information processing by
directly communicating between them via chemical signals
such as ATP, Ca2+ and leukemia inhibitory factors (LIF) for
myelination (Ishibashi et al., 2006; Fields and StevensGraham, 2002). Since non-neuronal cells in the nervous system
function in diverse ways, further investigation of Cdc2 in this
system could provide insight into the regulatory mechanism of
nerve regeneration.
Materials and Methods
Rats and sciatic nerve surgery
The sciatic nerve of Sprague-Dawley rats (7-8 weeks old, male) was exposed on
the middle thigh and crush injury on the nerve was given by holding twice with
forceps for 30 seconds, as described previously (Seo et al., 2006). All procedures
were in strict accordance with the NIH guide for the care and use of laboratory
animals and approved by the Committee on Use of Live Animals for Teaching and
Research at Daejeon University. Nerves were prepared 0-14 d.p.c. for western blot
analysis, immunohistochemistry, or primary cell culture.
Primary Schwann cell and DRG sensory neuron culture
For Schwann cell and DRG sensory neuron culture, sciatic nerve and DRG at lumbar
4-5 were dissociated by treatment with 125 U/ml type XI collagenase (Sigma, St
Louis, MO) in DMEM for 80 minutes at 37°C, and washed twice with DMEM.
Cells were treated with 0.5 mg/ml type SII trypsin for 15 minutes and followed by
inhibition reaction for 5 minutes in 1 mM EDTA, 100 ␮g/ml of soybean trypsin
inhibitor and 40 ␮g/ml of DNase I. Cells (1⫻106 cells per dish) were then plated
onto 12-mm coverslips (Bellco, Glass Inc. Vineland, NJ) precoated with 0.01%
poly-L-ornithine (Sigma, St Louis, MO) and laminin (0.02 mg/ml, Collaborative
Research, Bedford, MA). Cells were cultured for 12 hours, and changed to DMEM
containing 10% serum [5% fetal bovine serum (Gibco, Australia) plus 5% horse
Sciatic nerve with or without crush injury was placed in ice-cold DMEM, and placed
on 12-mm coverslips precoated with a mixture of poly-L-ornithine and laminin.
Tissue was incubated in 200 ␮l culture medium (DMEM containing 5% fetal bovine
serum, 5% horse serum and 2 mM glutamine and 1% penicillin-streptomycin) for
1 hour and supplemented with 300 ␮l medium. The explant culture was replaced
with 500 ␮l of fresh medium 16 hours later and incubated for 48 hours. Then the
culture was fixed with 4% paraformaldehyde/4% sucrose for immunofluorescence
staining with anti-S100␤ antibody (1:200, Dako) and anti-Cdc2 antibody (1:100,
p34, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA). Average
migration distance in each explant (30-100 cells per explant) was determined by
measuring the distance between explant border and stained Schwann cells.
Recombinant DNA construct, transfection and infection
Recombinant pRcCMV5 plasmids encoding wild-type (wt) caldesmon or 7th
mutant caldesmon in which all seven Cdc2 phosphorylation sites were replaced with
Ala were kindly provided by S. Matsumura (Rutgers University, Piscataway, NJ).
pRcCMV5-Cdc2wt-HA and pRcCMV5-Cdc2dn-HA were from Sander van den
Heuvel (Harvard University, Boston, MA). Transient transfection into Schwann
cells (1⫻104 cells per 24-well plate) was performed using 3 ␮g pCMV5-Cdc2wtHA, pCMV5-Cdc2dn-HA, pRcCMV-caldesmon wt, pRcCMV-7th caldesmon or
pCMV5 together with 1 ␮g pmaxGFP plasmid, by the calcium phosphate protocol
as described (Namgung and Xia, 2000). With this procedure, cotransfection
efficiency was routinely higher than 70%. Cells were harvested 48 hours later. For
migration analysis, 1⫻104 cells were plated on the filter chambers (Costar) and
harvested 2 days later for counting GFP-positive cells on the coverslip. Adenoviral
vector (Ad) expressing HA-tagged Cdc2 wt or Cdc2 dn cDNA was prepared using
AdEasy system (Stratagene, La Jolla, CA). Ad-Cdc2 DNA was subcloned into
pShuttle-CMV vector and cotransformed into E. coli BJ5183 cells with Adeasy-1
vector. Selected recombinant plasmids were transfected into HEK293 cells and
replication-deficient recombinant adenoviruses were generated. Adenoviruses
containing Cdc2 were isolated using a commercially available adenovirus
purification kit (Cell Biolabs, San Diego, CA) following the manufacturer’s
instructions. Purified viruses were aliquoted and stored at –80°C. Viral titer (PFU
per milliliter: 1⫻1011) was determined using the agarose overlay method (Becker
et al., 1994).
Transplantation of infected Schwann cells into sciatic nerve
Schwann cells (1⫻104) were mixed with 40 ␮l adenoviral stock solution and
harvested 2 days later. Infected Schwann cell suspension (1⫻104 cells in 5 ␮l) was
injected at the injury site of the sciatic nerve using the microsyringe (Microliter
Spritze, Innovative Labor System, Germany). An average of 75% of the Schwann
cells used for transplantation expressed GFP, and 59% and 63% of the GFP-positive
cells expressed wt-Cdc2 and dn-Cdc2 in viral constructs respectively when HAtagged Cdc2 expression was analyzed by immunofluorescence staining with antiHA antibody (1:500, Santa Cruz Biotechnology). The implanted cells were allowed
Cdc2-mediated Schwann cell migration
253
Journal of Cell Science
Fig. 7. Effect of Cdc2 activity on neurite outgrowth
of DRG sensory neurons. (A) Representative images
of neurite outgrowth of DRG sensory neurons under
different culture conditions. Dissociated cells were
prepared from DRGs (lumbar 4-5) and sciatic nerves
of animals that had or had not been subjected to
preconditioning injury in the sciatic nerves (7 d.p.c.).
Pre, injury preconditioned; Non-pre, nonpreconditioned; SN, sciatic nerve. The primary
sensory neurons and Schwann cells so derived were
co-cultured. Cells were visualized by NF-200
immunostaining. (B) Quantification of neurite
outgrowth of DRG sensory neurons. The number of
neurite branch points in DRG sensory neurons cocultured with Schwann cells of the sciatic nerve was
significantly decreased by 10 ␮M roscovitine
treatment. (**P<0.01; n=4). Bar, 50 ␮m.
to migrate along the nerve for 3 or 7 days, and nerve sections (20 ␮m thickness)
were prepared for immunofluorescence staining with anti-Neurofilament 200 (NF200) antibody (1:200, rabbit, Sigma). For quantitative analysis on migrated cells,
images of GFP-positive cells in the nerve sections were captured by a digital camera,
and transferred to Photoshop. The numbers of infected Schwann cells in a defined
area (470 ␮m ⫻ 590 ␮m) 0 mm, 1.5 mm, 3 mm, 5 mm and 7 mm from the injury
site were counted.
RT-PCR
Total RNA was extracted from sciatic nerve by Easy Blue (Intron Biotechnology,
Korea), and was used for reverse transcriptase reaction (MMLV RT, Promega,
Madison, WI) for 1 hour at 37°C, followed by PCR amplification of Cdc2 or actin
DNA for 30 cycles using Taq DNA polymerase (Takara, Japan). RT-PCR was
performed as suggested by the manufacturer. The primer sequences for Cdc2 were
5⬘-ATCGGAGAAGGGACTTATGG-3⬘ as a forward primer and 5⬘TGCAGGGATCTACTTCTGG-3⬘ as a reverse primer, and the sequences for actin
were 5⬘-CACACTGTGCCCATCTATGA-3⬘ as a forward primer and (5⬘TACGGATGTCAACGTCACAC-3⬘) as a reverse primer. The amplified PCR
products were 677 bp and 409 bp for Cdc2 and actin respectively.
Fluorescence in situ hybridization (FISH)
Cross sections (20-␮m thick) of sciatic nerves cut on a cryostat and mounted on
positively charged Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA) were
fixed with 4% paraformaldehyde for 10 minutes, rinsed twice in 0.1% DEPC water,
washed in 0.1 M triethanolamine (TEA, pH 8.0), and treated with 0.25% acetic
anhydride in 0.1 M TEA for 10 minutes. After washing twice in 2⫻ SSC, sections
were dehydrated in an ethanol series (50%, 70%, 90%), and hybridized overnight
at 58°C using 3 ng/␮l fluorescein-UTP (Roche, Germany)-labeled Cdc2 riboprobes
in hybridization buffer (0.3 M NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 50%
formamide, 1⫻ Denhardt’s, 10% dextran sulphate, 10 mM DTT). pcDNA3-Cdc2
was linearized with HindIII and EcoRI digestion for anti-sense and sense probe
preparation respectively. Sense and anti-sense RNA probes were prepared by
fluorescence labeling reaction with RNA polymerase SP6 and T7 respectively as
suggested by the manufacturer (Promega, Madison, WI). After hybridization, slides
were rinsed for 10 minutes in 2⫻ SSC containing 10 mM ␤-mercaptoethanol and
1 mM EDTA, washed for 30 minutes in RNase solution (0.5 M NaCl, 10 mM TrisHCl pH 8.0, 20 ␮g/ml RNase), then washed for 10 minutes with 2⫻ SSC. After
incubating for 2 hours at 60°C in 0.1⫻ SSC containing 10 mM ␤-mercaptoethanol
and 1 mM EDTA, sections were rinsed in 0.5⫻ SSC and dehydrated in an ethanol
series (50%, 70%, 90% containing 0.3 M ammonium acetate). Sections after
hybridization reaction were permeabilized in 0.5% nonidet P-40 solution and
subsequently treated for immunofluorescence staining with anti-S100␤ primary
antibody (1:200, Dako) and Rhodamine-goat anti-rabbit secondary antibody (1:400;
Molecular Probes, Eugene, OR) (see below). Fluorescence images were analyzed
by confocal laser-scanning microscopy (LSM 510, Carl Zeiss, Germany).
Western blotting and kinase assay
Nerve segments or cultured Schwann cells in 50-200 ␮l of Triton lysis buffer (20
mM Tris-HCl, pH 7.4, 137 mM NaCl, 25 mM ␤-glycerophosphate, pH 7.14, 2 mM
sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 10%
glycerol, 5 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, 3 ␮M benzamidine, 0.5 mM DTT,
1 mM PMSF) were sonicated, and the supernatant was removed after centrifugation
at 14,000 g for 10 minutes at 4°C. Antibodies used are: anti-Cdc2 antibody (1:1000,
p34, mouse monoclonal, Santa Cruz Biotechnology), anti-phospho-caldesmon
antibody (1:1000, rabbit polyclonal, Santa Cruz Biotechnology), anti-actin antibody
(1:5000, clone no.C4, mouse monoclonal, MP Biomedicals, Aurora, OH), and
horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000, goat antirabbit; Santa Cruz Biotechnology, or sheep anti-mouse; Amersham Biosciences,
France). Protein (10 ␮g) was used for western analysis as described previously (Seo
et al., 2006).
Cdc2 kinase assay was performed as described previously with some
modifications (Namgung and Xia, 2000; Qiao et al., 2006). Anti-actin antibody (3
254
Journal of Cell Science 120 (2)
␮l; clone no. C4, mouse monoclonal, MP Biomedicals, Aurora, OH) was incubated
in 250 ␮l PBS containing 8% protein A Sepharose for 16 hours at 4°C and beads
were washed once with cold Triton lysis buffer. Cell lysate (200 ␮g of protein in
100 ␮l reaction) was immunoprecipitated with anti-actin antibody conjugated to
protein A Sepharose for 3 hours at 4°C. The beads were washed twice each with
Triton lysis buffer and by cold kinase buffer (25 mM HEPES, pH 7.4, 25 mM ␤glycerophospohate, pH 7.14, 25 mM MgCl2, 0.1 mM Na3VO4, 0.5 mM DTT). The
immunocomplexes were precipitated by brief centrifugation (1500 g, 1 minute) and
incubated with 30 ␮l of kinase reaction buffer containing 1 nmole ATP, 10 ␮Ci of
[␥-32P]ATP (3000 ␮Ci/ml, Amersham), and 1 unit of Cdc2 recombinant kinase
(New England Biolabs, Beverly, MA) for 30 minutes at 30°C. In the control
reaction, 2 ␮g histone H1 or 10 ␮M roscovitine was included in the reaction
mixture. Samples were boiled, and the supernatants were resolved by 10% SDSPAGE. The gels were dried and analyzed by autoradiography. For the purpose of
western analysis of kinase reaction products, the immunocomplexes were incubated
with kinase reaction mixture including 1 nmole ATP and 1 unit of Cdc2 recombinant
kinase, and were used for western blot analysis using anti-phospho-caldesmon
antibody (1:1000, rabbit polyclonal, Santa Cruz Biotechnology, Inc.).
Journal of Cell Science
Immunofluorescence staining
Nerve sections or cultured cells were fixed with 4% paraformaldehyde and 4%
sucrose in PBS at room temperature for 40 minutes, permeabilized with 0.5%
nonidet P-40 in PBS, and blocked with 2.5% horse serum and 2.5% bovine serum
albumin for 4 hours at room temperature. The staining reaction was performed by
incubating with primary antibodies including anti-Cdc2 antibody (1:100, p34,
mouse monoclonal, Santa Cruz Biotechnology), anti-S100␤ antibody (1:200,
Dako), anti-caldesmon antibody (1:200, mouse monoclonal Sigma), anti-phosphocaldesmon antibody (1:200, rabbit polyclonal, Santa Cruz Biotechnology), antineurofilament 200 antibody (1:200, rabbit, Sigma), and anti-␤III-tubulin antibody
(1:200, TUJ1, rabbit polyclonal, Covance, Berkeley, CA), followed by Fluoresceinlabeled goat anti-mouse (1:400; Molecular Probes) or Rhodamine-labelled goat
anti-rabbit secondary antibodies (1:400; Molecular Probes) in 2.5% horse serum and
2.5% bovine serum albumin for 1 hour at room temperature. Cellular nuclei were
stained with 2.5 ␮g/ml of Hoechst 33258 (bis-benzimide, Sigma) for 10 minutes
before the final washing with 0.1% Triton X-100 in PBS, and the sections or cells
were coverslipped with gelatin mount medium. We always included control sections
treated with secondary antibody alone, which usually did not have any visible
images. In cases where the nonspecific signals were high, data from the same
experiments were not analyzed further. Samples were viewed with a Nikon
fluorescence microscope, and the images were captured using Nikon camera. The
merged images were produced using layer-blending mode options of Adobe
Photoshop (Version 5.5).
Statistical analysis
Data were presented as mean ± s.e.m. The mean numbers of data in individual
groups were compared by one-way ANOVA followed by Tukey test (SPSS
computer software version 12.0), and statistically significant differences were
reported as *P<0.05, **P<0.01, ***P<0.001.
We are grateful to S. Matsumura (Rutgers University) and Sander
van den Heuvel (Harvard Medical School) for providing the plasmids
and David Elzi (University of Washington) for critical reading of the
manuscript. This work was supported by the Korea Research
Foundation (C00180 and C00161).
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