Download Thrombin Activation of S-Phase Reentry by Cultured

Experimental Cell Research 281, 101–106 (2002)
doi:10.1006/excr.2002.5650
Thrombin Activation of S-Phase Reentry by C ultured Pigmented
E pithelial C ells of Adult N ewt Iris
András Simon 1,2 and Jeremy P. Brockes
Department of Biochemistry and Molecular Biology, University College London, London, WC1E 6BT United Kingdom;
and Medical Nobel Institute, Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden
Following local injury or tissue removal, regeneration in urodele amphibians appears to be dependent
on cell cycle reentry and dedifferentiation of postmitotic, terminally differentiated cells in the remaining
tissues. Regeneration of the lens of the eye occurs by
the dedifferentiation of pigmented epithelial cells
(PEC) of the iris and their subsequent transdifferentiation into lens cells. A key question is how cell cycle
reentry is regulated. Here we demonstrate that thrombin activates S-phase reentry of newt PEC in vitro.
Based on these findings, and on previous experiments
showing that newt skeletal myotubes reenter the cell
cycle following thrombin stimulation, we suggest that
thrombin is a critical signal for initiation of vertebrate
regeneration. © 2002 Elsevier Science (USA)
INTRODUCTION
Urodele amphibians, such as newts and axolotls,
have a unique capacity among vertebrates to regenerate their body parts as adults. While loss or degeneration of the neuroretina and other ocular structures leads to visual handicap and eventually
blindness in most vertebrates, a newt can regenerate
its lens and neuroretina. Other newt structures
which can be replaced after injury include the upper
and lower jaws, spinal cord, parts of the central
nervous system, the cardiac musculature, and the
intestine [1, 2].
Several studies emphasize the role of the plasticity of
the differentiated state during amphibian regeneration and show that the progenitor cells of the regenerate are derived from postmitotic, differentiated cell
types (for a review see [3]). Multinucleated skeletal
muscle cells reenter the cell cycle and undergo fragmentation into proliferating, cycling progeny cells during limb and tail regeneration in urodeles. These prog1
Present address: Medical Nobel Institute, Department of Cell
and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
2
To whom correspondence and reprint requests should be addressed. Fax: 46(0)8308374. E-mail: [email protected].
eny cells contribute to the growth zone or blastema,
from which the cells of the regenerate originate [4 – 8].
Similarly, regeneration of the heart depends on cell
cycle reentry and proliferation of cardiomyocytes in the
vicinity of the injury [9]. Regeneration of ocular structures such as the lens and neuroretina depends on
transdifferentiation. Upon removal of the lens, pigmented epithelial cells (PEC) in the dorsal margin of
the iris enter S phase, lose their pigmentation, and give
rise to a new lens. Transdifferentiation to lens is confined normally to PEC of the pupillary margin of the
dorsal iris and the new lens does not arise from the
ventral iris [10].
The molecular mechanisms that induce cells to
leave the postmitotic arrest have long remained
enigmatic. Recently it was shown that the postmitotic arrest of newt skeletal myotubes in culture can
be undermined by stimulating them with serum [11].
The active component in serum is not a known protein growth factor but an as yet unidentified molecule, which is activated by thrombin but which is
distinct from the protease [12]. Thus, a subthreshold
concentration of serum can be activated by digestion
with thrombin, followed by inhibition of residual protease activity. The thrombin-derived activity, which
leads to cell cycle reentry of the newt myotubes, is
inactive on mouse myotubes, although it is present
in sera from various mammalian sources [12, 13]. It
is appealing to speculate that following injury, the
activation of thrombin generates a signal, which
newt cells can transduce and which evokes a key
cellular response in regeneration.
Here we have addressed the question of whether
the thrombin-derived activity may represent a common signal for cells involved in a different context of
regeneration. Since cell cycle reentry of PEC is the
first step in lens regeneration, we established a culture model in which the PEC are quiescent but respond to appropriate molecules that can induce cell
cycle reentry. We found that the thrombin-derived
activity is able to induce S-phase reentry of PEC in
vitro.
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© 2002 Elsevier Science (USA)
All rights reserved.
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SIMON AND BROCKES
FIG. 1. Morphology of PEC in culture. (A and D) Phase-contrast image of PEC at 20- and 40-fold magnifications, respectively. (B and E)
DAPI (blue) staining of the nuclei at 20- and 40-fold magnifications, respectively. (C and F) BrdU staining of nuclei in S-phase (red) at 20and 40-fold magnifications, respectively. Scale bar, 50 !m
THROMBIN AND S-PHASE REENTRY
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FIG. 2. S-phase reentry of PEC induced by increasing concentration of FCS. Bars represent the averages of three independent wells in
a representative experiment. Dots indicate the results in the individual wells.
MATERIAL AND METHODS
Animals. Red spotted newts, Notophthalmus viridescens, were
purchased from Charles Sullivan Co. (TN). Animals were kept in tap
water at 19°C and fed once a week.
Preparation of PEC. Animals were anesthetized by immersion in
0.1% MS 222 (Sigma) dissolved in tap water. The eyeballs were
removed and collected in a tissue culture dish containing PBS adjusted to amphibian osmolarity. The eyeballs were washed in 70%
ethanol for 2 min and transferred to Leibowitz-15 (L-15) medium
(Gibco), adjusted to amphibian osmolarity, and supplemented with
0.5% bovine serum albumin (BSA), penicillin/streptomycin, and Lglutamine. Corneal cells were removed and an anterior eye cup was
created by cutting the eyeball along the iris. The lens was removed
and the iris rings were placed in L-15 medium containing 0.5% BSA
and 0.5% dispase grade II (La Roche). If dorsal and ventral cells were
analyzed separately, the iris rings were cut into dorsal and ventral
halves at this stage. The tissues were incubated at 25°C for 4 h and
the sheet of PEC was separated from the underlying stromal cells.
Dissociation of the PEC sheet was achieved by incubation with 0.25%
trypsin–EDTA solution for 10 min at room temperature. Trypsin was
diluted 1/20 in L-15 medium containing 0.5% BSA, and the cells were
centrifuged at 100g for 5 min. Cells were resuspended in L-15 medium containing 0.5% BSA, and approximately 600 cells were plated
onto collagen type IV- (Sigma) coated wells in 96-well plates. Thirty
to fifty percent of the plated cells attached and spread. With this
method over 99% of the cells were PEC.
S-phase reentry assay. Seven days after plating, the cells were
shifted to L-15 medium containing various concentrations of fetal
calf serum (FCS; Gibco; Lot F40F8814) with or without thrombin or
additional growth factors. After 3 days, bromodeoxyuridine (BrdU;
La Roche) was added to a final concentration of 10 !M. After 18 –20
h the cells were fixed for 30 s in 2% paraformaldehyde and postfixed
for 5 min in ice-cold methanol. To test the effect of growth factors,
medium containing 0.25% FCS was supplemented with 40 ng/ml
purified mouse EGF (Collaborative Research), human recombinant
FGF-2 (Gibco; both factors are kind gifts from Professor Jonas
Frisén), PDGF-C (a kind gift from Professor Ulf Eriksson), or all
three growth factors simultaneously. The effect of thrombin was
assayed in the presence of 0.25% FCS and 100 !g/ml crude preparation of bovine thrombin (Calbiochem). The effect of the thrombinderived activity was analyzed essentially as described in [12].
Briefly, medium containing 1% FCS was incubated with purified
bovine thrombin (Enzyme Research Laboratories) for 24 h at 25°C.
Thrombin was inactivated using a 50-fold molar excess of D-Phe-ProArg chloromethyl ketone (PPACK; Sigma). The activated medium
containing 1% FCS was diluted to a final concentration of 0.25% FCS
and added to cells. In control experiments PPACK and purified
thrombin were added simultaneously in order to prevent activation.
Thrombin activity was measured spectrophotometrically using Chromosyme TH (La Roche) as substrate according to the manufacturer’s
recommendations. Thrombin activity was completely inhibited by
PPACK.
Immunocytochemistry and image analysis. BrdU staining was
performed as described [11] and visualized using Alexa 546-conjugated anti-mouse IgG-specific secondary antibody (Molecular
Probes). The nuclei were stained by DAPI. Cells were observed using
a Nikon inverted microscope and pictures were captured by a color
CCD camera.
Data analysis. The data represent the average of results from
three independent wells in a representative experiment. Between 90
and 300 cells were counted in each well.
RESULTS AND DISCUSSION
We first determined the conditions under which PEC
are proliferating or quiescent. PEC were prepared and
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FIG. 3. Thrombin induces S-phase reentry of PEC. “Crude thr” indicates that medium containing 0.25% FCS was supplemented with a
crude preparation of bovine thrombin as described under Material and Methods. “0.25% FCS non-act” indicates that medium containing
0.25% FCS was preincubated with both purified bovine thrombin and thrombin inhibitor for 24 h before addition to cells. “0.25% FCS act”
indicates that medium containing 0.25% FCS was preincubated with purified thrombin for 24 h and thrombin activity was irreversibly
inhibited before addition to cells. Bars represent the average of three independent wells in a representative experiment. Dots indicate the
results in the individual wells.
plated in serum-free medium. Figure 1 shows the morphology of the cells at two different magnifications. The
cells are heavily pigmented, often display a hexagonal
shape with clearly visible nuclei, and tend to attach to
each other and to form islets (Figs. 1A, 1B, 1D, and 1E).
In the presence of 10% FCS a substantial portion of
PEC reenter S phase as assayed by BrdU incorporation
(Figs. 1C and 1F). Most of the cells are quiescent in
serum-free medium and as little as 1% FCS induced
DNA replication in more than 20% of PEC (Fig. 2). The
maximal level of S-phase reentry was observed in the
presence of 10% FCS (Fig. 2). It should be noted that
different sources of serum resulted in different levels of
S-phase reentry (data not shown), although the overall
pattern was the same as shown in Fig. 2. We therefore
performed all experiments in the same batch of serum,
as specified under Material and Methods.
Next we asked whether thrombin could induce Sphase reentry as observed in the case of newt skeletal
myotubes. We assayed the effect of a crude preparation
of bovine thrombin in the presence of 0.25% FCS, since
the background level at this serum concentration was
relatively low. Crude thrombin resulted in a 6.5-fold
increase in BrdU-positive cells compared to the control
(Fig. 3). To test whether this effect of thrombin was
direct or indirect, we activated FCS-containing medium with purified thrombin and inactivated the activity of the protease irreversibly by PPACK before addition to the cells [12]. As a control we used medium
which was preincubated with thrombin in the presence
of thrombin inhibitor. As shown in Fig. 3, thrombinactivated serum induced a 5.5-fold increase in the
number of PEC that entered S-phase. The crude preparation of thrombin also induced S-phase reentry in
serum-free medium, but to a lower extent compared to
activation in the presence of serum (data not shown),
suggesting that crude thrombin contains the thrombinderived activity identified by Tanaka et al. [12].
Lens regeneration in situ is dependent on the PEC of
the dorsal margin of the iris, while ventral PEC do not
participate in formation of the new lens [14]. To test
whether this difference is correlated to thrombin responsiveness, we separated the iris into dorsal and
ventral halves prior to the removal of PEC and seeded
dorsal and ventral cells in separate wells. We did not
find any difference between dorsal and ventral cells
with respect to responsiveness to subthreshold concentration of serum, which was activated with pure throm-
THROMBIN AND S-PHASE REENTRY
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FIG. 4. Both dorsal and ventral PEC reenter S phase upon stimulation with thrombin-activated serum. Labels should be interpreted as in Fig.
3. Bars represent the average of three independent wells in a representative experiment. Dots indicate the results in the individual wells.
FIG. 5. Growth factors induce S-phase reentry of PEC. “GF-MIX” indicates that all three growth factors were added to the medium. Bars
represent the average of three independent wells in a representative experiment. Dots indicate the results in the individual wells.
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bin. Dorsal and ventral cells showed identical responses compared to control (Fig. 4). These results are
in agreement with previous results by Eguchi et al.
[15], which showed that both dorsal and ventral cells
form lentoid bodies in vitro, despite the fact that only
dorsal cells participate in lens regeneration in vivo.
As serum also contains mitogenic growth factors, we
wanted to see whether purified growth factors could
induce S-phase reentry of PEC. We tested three different growth factors, EGF, PDGF-C, and FGF-2. All
three induced cell cycle reentry to approximately the
same level either alone (PDGF-C, 9.4-fold; FGF-2, 11fold; EGF, 10.7-fold), or in combination (10.5-fold), suggesting that PEC express receptors for these growth
factors (Fig. 5).
The phenomenon of Wolffian regeneration [16] is a
demonstration of the reprogramming and transdifferentiation of a fully differentiated cell type, the PEC.
Transdifferentiation begins with cell cycle reentry, and
our results show that PEC respond to thrombin stimulation in culture in the same way as do skeletal myotubes. While the implications of this finding would still
require extension from in vivo experiments, our results
indicate that the thrombin-activated pathway may
represent a common signal for postmitotic cells, which
are reactivated during regeneration in newts. Various
growth factors in addition to thrombin evoke DNA
replication in PEC. This contrasts with the case of
newt skeletal myotubes, which are refractory to growth
factor stimulation [11, 12]. One simple interpretation
of these data could be that PEC have receptors for
these growth factors, whereas the myotubes have receptors for only the thrombin-activated factor. Nonetheless, these observations are in agreement with several previous studies which underline the role of
mitogenic growth factors in proliferation and transdifferentiation of PEC to lens or lentoid bodies [17–19].
The culture system described here provides an opportunity for systematic analysis of the proliferative
potential of PEC under different conditions. It may be
useful in future experiments to investigate how cell
cycle reentry and transdifferentiation are coupled to
each other and how specific agents may influence their
course. One important task for the future is also to
determine whether thrombin is able to induce cells to
complete the mitotic cycle.
We are most grateful to Y. Imokawa for help with initial preparation of PEC and for helpful discussions. We thank K. Agata for advice
on culture conditions, E. Tanaka for suggestions, S. Sandberg for
comments on the manuscript, and J. Frisén and U. Eriksson for
providing growth factors. The financial support from an MRC proReceived June 18, 2002
Revised version received August 15, 2002
Published online October 11, 2002
gram grant to J.P.B. and to A.S. from The Wenner–Gren Foundation,
The Swedish Research Council, Carl Tryggers Stiftelse, Åke Wibergs
Stiftelse, Magnus Bergvalls Stiftelse, and Stiftelsen Lars Hiertas
Minne is greatly appreciated.
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