From the Division of Experimental Hematology, Children's Hospital Research Foundation, University of Cincinnati, Cincinnati, Ohio 45229
Received for publication, January 13, 2003, and in revised form, February 4, 2003
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ABSTRACT |
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The p19Arf-p53 tumor suppressor
pathway plays a critical role in cell-cycle checkpoint control and
apoptosis, whereas Rho family small GTPases are key regulators of actin
structure and cell motility. By using primary mouse embryonic
fibroblasts that lack Arf, p53, or both, we
studied the involvement of the p19Arf-p53 pathway in the
regulation of cell motility and its relationship with Rho GTPases.
Deletion of Arf and/or p53 led to actin
cytoskeleton reorganization and a significant increase in cell
motility. The endogenous phosphoinositide (PI) 3- kinase and Rac1
activities were elevated in Arf The p53 and pRb cell cycle inhibitors and their regulators,
including p19Arf and p27Kip1, are well
established tumor suppressors that are components of a complex
signaling network central to tumor suppression (1). Deletion or
mutation in these tumor suppressors or their regulators occurs in the
majority of tumor cases and correlates with the onset of a wide
spectrum of cancer (2). Specifically the p19Arf-p53 tumor
suppressor pathway is thought to be involved primarily in monitoring
proliferation signals to prevent cells from uncontrolled growth (3).
Rho family small GTPases, on the other hand, are molecular switches
that transduce diverse intracellular signals leading to cell
proliferation, gene induction, apoptosis, and in particular,
cytoskeleton remodeling and migration (4). Accumulating evidence has
implicated the Rho GTPases, RhoA, Rac1, and Cdc42, in many aspects of
tumor development, including tumor cell migration and proliferation
(5). Many mitogenic signals, including those from growth factor
receptors and integrins, can promote the exchange of GDP for GTP on Rho
GTPases, enabling them to interact with an array of effector targets to
elicit cytoskeletal changes (6, 7).
Although much of the attention in tumor suppressor studies has been
focused on their roles in cell cycle control and/or apoptosis, preliminary evidence has become available suggesting a potential functional linkage between p53 and cell morphological effect/cell motility. For example, p53 has been shown to regulate the transcription of a number of genes involved in cell morphology and/or movement, including that of smooth muscle To investigate the functional connection between the tumor suppressors
and Rho GTPase-regulated cytoskeleton events, we have examined the
actin cytoskeletal structure and migration capability of
p19Arf-, p53-, p27Kip1-, or pRb-deficient
primary mouse embryonic fibroblast
(MEF)1 cells. We report that
the p19Arf-p53 tumor suppressor pathway, but not the
related p27Kip1-pRb pathway, negatively regulates cell
motility through suppression of the PI 3-kinase-Rac1 signaling module.
DNA Constructs--
Site-directed mutagenesis of Rac1, Cdc42,
RhoA, and p53 was performed by the polymerase chain reaction-based,
oligonucleotide-mediated method (16). The retroviral constructs
expressing p19Arf, p53 wild type or mutant, and dominant
negative Rho GTPases (Rac1N17, Cdc42N17, and RhoAN19) were generated by
ligating the corresponding cDNA fragments into the BamHI
and EcoRI sites of the retroviral GFP bicistronic vector,
MIEG3 (17).
Cell Culture, Retroviral Infection, and Cell
Imaging--
Primary MEFs from p19Arf
Primary MEFs were seeded onto coverslips at 3 × 104
cells/slip density. The cells were serum-starved for 12 h prior to
fixation. The F-actin structure of the cells was visualized by
rhodamine-phalloidin staining, and the stained cells were analyzed by
using a conventional fluorescence microscope (Zeiss).
Cell Motility and Migration Assays--
Single cell motility
assays using the colloidal gold particles were carried out as described
(19). The wound healing assays to semi-quantitatively determine the
migration distance were performed following a scratch of the confluent
monolayer of cells as described (20). The migration distances of cells
were monitored at various times and were measured at 9 h after the
introduction of the wound under low serum (0.5%) conditions.
PI 3-kinase Assay, Chemical Inhibitors, and
Immunoblotting--
The endogenous PI 3-kinase activities of
Arf
Prior to Western blotting, the protein contents of the cell lysates
were normalized by the Bradford method.
Anti-phospho-Akt(Ser473) and anti-PTEN
antibodies were obtained from Cell Signaling, Inc., anti-p85 and the
anti-p110 of PI 3-kinase antibodies were obtained from Upstate
Biotechnology, Inc., and the PI 3-kinase inhibitors wortmannin and
LY294002 were obtained from Sigma.
Endogenous Rho GTPases Activity Assay--
GST-PAK1,
GST-Rhotekin, and GST-WASP, which contain Rac1, RhoA, and the
Cdc42- interactive domains of PAK1, Rhotekin, and WASP, respectively,
were used to probe the endogenous Rac1-GTP, RhoA-GTP, and Cdc42-GTP
activities as described (20).
p19Arf, p53, p27Kip1, and pRb are well
recognized cell cycle regulators that are critical to checkpoint
control. p19Arf, in response to oncogenic signals,
stabilizes p53 by sequestering Mdm2, a negative regulator of p53 (3),
whereas p27Kip1, on its way through pRb, regulates
G1 transition by modulating growth-essential E2F
transcription factors (1). All four are important components of a
complex tumor suppressor network. To examine their potential
involvement in the regulation of cell actin structure and motility, we
have generated the p19Arf-, p53-, p27Kip1-, or
pRb-deficient primary MEFs from the respective tumor suppressor knock-out mice. To avoid possible alteration of cell properties under
tissue culture conditions, only low passages (<6 passages) of the
primary cells were used and at least two independent p53- or
p19Arf-deficient MEF preparations were examined.
In subconfluent culture and serum-free conditions, a significant
percentage of the p19Arf- or p53-deficient cells appeared
to be in a round shape and displayed intense cortical actin staining,
whereas the p27Kip1- or pRb-deficient cells were similar to
wild type MEFs in morphology and actin structure (Fig.
1A). To determine whether the
altered actin structures might be associated with cell motility
modulation, the cells were plated onto colloidal gold-coated plates,
and the motility of single cells was traced by the trails left behind their movement paths. As shown in Fig. 1B, in the absence of
serum stimulation, both p19Arf- and p53-deficient cells
produced long trails, whereas the Kip1 and Rb
knock-out cells were similar to the wild type cells, showing no sign of
movement.
/
and
p53
/
cells, and these activities are
required for p19Arf- and p53-regulated migration.
Reintroduction of the wild type Arf or p53
genes into Arf
/
or
p53
/
cells reversed the PI 3-kinase and Rho
GTPase activities as well as the migration phenotype. These
results suggest a functional relationship between an established tumor
suppressor pathway and a signaling module that controls actin structure
and cell motility and show that p19Arf and p53 negatively
regulate cell migration by suppression of PI 3-kinase and Rac1 activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-actin (8), collagens II
1 and
VI
1 (9), vascular epidermal growth factor (10),
metalloproteinase-1 (11), and fibronectin (12). Moreover,
overexpression of p53 seems to cause a partial reversion of Ras-induced
morphological transformation (13) and to inhibit cell migration (14). A recent study further suggests that p53 may have a role in
Cdc42-mediated filopodia formation and cell polarization (15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
,
p53
/
, p19Arf
/
/p53
/
,
p27Kip1
/
, and pRb
/
mice (all of the
C57BL/6 × 129/sv background) were generous gifts from Dr. Martine
Roussel (St. Jude, Memphis, TN) and were generated as described
previously (18). Recombinant retroviruses were produced using the
ecotropic Phoenix packaging cell system (17). The primary MEFs were
infected with the retroviruses and harvested 48-72 h after infection.
GFP-positive cells were isolated by fluorescence-activated cell sorting.
/
, p53
/
,
Arf
/
/p53
/
, and
Arf
/
or p53
/
cells reconstituted with Arf or p53 were assayed
according to a described protocol (21). Briefly the cells were lysed in
buffer A containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.3 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1%
Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mM sodium orthovanadate and 25 mM NaF.
After centrifugation at 4 °C for 30 min at 14,000 rpm, the protein
contents in the supernatant of cell lysates were measured by the
Bradford method. An equal amount of proteins was incubated with
anti-p85 polyclonal antibody coupled to protein A-agarose (Upstate
Biotechnology) overnight or subjected to anti-p85 or anti-p110 Western
blot analysis. The immunoprecipitates were washed twice with buffer C
containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl,
and 10 mM MgCl2 and washed once with a kinase
assay buffer (20 mM Tris-HCl, pH 7.6, 10 mM
MgCl2). 5 µg of sonicated phosphatidylinositol together
with [
-32P]ATP (200 µCi/ml) in 45 µl of the kinase
assay buffer were incubated with the washed beads at 25 °C for 10 min. The reactions were terminated by the addition of 100 µl of 1 N HCl. The reaction products were extracted by 200 µl of
CHCl3/MeOH (1:1) and resolved on a TLC silica plate coated
with potassium oxalate in a solvent containing
CHCl3/MeOH/NH4OH, 4 M (9:7:2). The
PI 3-kinase reactions were analyzed by autoradiography.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The effect of the deletion of p19Arf, p53,
p27, or Rb on actin structure and cell motility of primary MEF
cells. 2×104 of wild type (WT) or
knock-out MEF cells (Arf /
,
p53
/
, p27
/
,
Rb
/
) were cultured overnight on coverglass.
Subconfluent cells were starved in medium containing 0.5% serum for
12 h. After fixation in 3.7% formaldehyde, cells were
permeabilized with 0.1% Triton X-100 and stained for F-actin by using
rhodamine-conjugated phalloidin (A). Single cell-based
motility was observed 9 h after the cells were plated on colloidal
gold-coated culture dishes (B).
To further quantify cell migration rate, we measured the migration
distance of these cells in a wound healing assay. The
Arf/
and p53
/
cells migrated about 2-fold faster than the wild type,
Kip1
/
, or Rb
/
cells at 9-h intervals under low serum (0.5% fetal bovine serum) conditions (Fig. 2, A and
B), at which time the difference in cell growth was less
than 10%. Similar results were also obtained when the cells were
serum-starved over a 16-h period (data not shown). When
p19Arf and p53 were reintroduced into
Arf
/
and p53
/
cells, respectively, by retroviral induction, the migration phenotype of these cells was fully reversed (Fig. 2, C and
D). The expression levels of p19Arf or p53 in the
reconstituted cells were comparable with or slightly higher than that
of the respective wild type cells (Fig. 2E). Thus,
p19Arf and p53 negatively regulate cell migration.
|
p19Arf and p53 display both interdependent and independent
activities in the regulation of cell cycle progression and in response to DNA damage (3). Deletion of both Arf and p53
alleles led to an equally mobile cell phenotype as
p19Arf/
or p53
/
cells in the single cell-based motility assay and in the wound healing assay (Fig. 3A).
Furthermore, introduction of p19Arf into the
Arf
/
/p53
/
cells
did not affect cell migration (Fig. 3A). It appears that p19Arf acts upstream of p53 in the regulation of cell
motility.
|
p53 functions as a DNA-binding dependent transcription factor in
mediating multiple gene activation that is critical to its tumor
suppressor activity (22). In addition, p53 is also known for a role in
negatively regulating the transcription of an array of genes by
repression of their transcription. To begin to address the issue of
what function of p53 is required for the regulation of cell motility,
we examined the ability of two p53 mutants, Gln22/Ser23 and His175, to suppress
the migration phenotype of p53/
cells.
Although both mutants are defective in transcriptional regulation of
p53-positive genes including that of p21Waf1/Cip1 (23-25),
the Gln22/Ser23 mutation is located in the
N-terminal transcription activation domain, which has also been shown
to be critical for the binding of TATA-binding protein-associated
factors (26, 27), whereas the His175 mutation resides in
the central DNA binding domain of p53, which is the site of a majority
of mutations seen in tumors (28). We observed that the
Gln22/Ser23 mutant was able to rescue the
migration phenotype of p53
/
cells similar to
wild type p53, but the His175 mutant was inactive (Fig.
3B). These results suggest that specific transcription
activity controlled by p53 is responsible for the regulation of cell
migration by the p19Arf-p53 tumor suppressor pathway.
PI 3-kinase has previously been implicated in the regulation of cell
migration (29). To investigate whether PI 3-kinase contributes to
p19Arf- and p53-regulated cell motility, the
endogenous PI 3-kinase activity and
phospho-Akt(Ser473) content, which is indicative of
the endogenous PI 3-kinase activation status of Arf
/
, p53
/
, and Arf
/
/p53
/
MEFs, were examined,
and the requirement of PI 3-kinase in p19Arf- and
p53-mediated cell migration was assessed by using specific pharmacological inhibitors wortmannin or LY294002 at defined
concentrations. As shown in Fig. 4,
A and B, genetic
deletion of Arf, p53, or Arf and p53
led to 3-5-fold increases in PI 3-kinase activity and
phospho-Akt(Ser473), which were readily reversed by the
reintroduction of Arf or p53. The expression
levels of p85
and p110, as well as that of Akt, were not altered in
these cells (Fig. 4B; data not shown). The enhanced PI
3-kinase activities are apparently required for the migration phenotype
observed in these cells, because inhibitors of PI 3-kinase
(wortmannin or LY294002) caused complete reversion of the migration
phenotype without affecting the basal movement of the cells (Fig.
4C). Under these conditions, wortmannin or LY294002 potently
inhibited PI 3-kinase activity (Fig. 4C and data not shown).
On the other hand, the expression level of PTEN, a PIP3
phosphatase that could be transiently regulated by p53 transcriptional
activity (30), was not altered in the Arf
/
,
p53
/
, or
Arf
/
/p53
/
cells
nor in Arf
/
or
p53
/
cells that were forced to express
Arf or p53 (Fig. 4D). We conclude that
Arf and/or p53 deletion up-regulates PI 3-kinase
activity, resulting in increased cell motility.
|
Rho family members Rac1, RhoA, and Cdc42 are downstream signaling
components of PI 3-kinase and have been implicated in the regulation of
cell migration (31). To determine whether these Rho proteins might be
involved in p19Arf- and p53-regulated migration, we
examined the activation status of these Rho proteins in Arf
/
and p53
/
cells by specific
effector domain pull-down assays. As shown in Fig.
5A, in the absence of serum
stimulation, the endogenous levels of GTP-bound active Rac1 and
RhoA in p19Arf
/
or
p53
/
cells increased by 2- and 6-fold,
respectively, compared with those in wild type cells, whereas Cdc42-GTP
increased by less than 20%. The Rac1, RhoA, or Cdc42 expression levels
were not affected by p19Arf or p53 deletion. When
p19Arf or p53 was reintroduced into the knock-out cells, the active Rho
protein contents were decreased to the level of wild type cells or
lower (Fig. 5A), indicating that p19Arf and p53
suppress the Rho protein activities. Furthermore, among the dominant
negative mutants of Rac1 (Rac1N17), RhoA (RhoAN19), and Cdc42
(Cdc42N17), only Rac1N17 was able to completely reverse the migration
phenotype in both Arf
/
and
p53
/
cells, reducing their migration rates
to that of the wild type MEFs, similar to the effect of reintroducing
the Arf or p53 gene (Fig. 5B). The
three Rho GTPase mutants were expressed equally well in the cells (Fig.
5B). These results indicate that Rac1 is a specific mediator
of p19Arf- and p53-regulated cell migration.
|
The p53 and Rb genes and their regulatory molecules are central to the growth malfunction of most tumor cells (1). The p19Arf-p53 pathway, specifically, constitutes a checkpoint in cell cycle progression that is responsive to the proliferative signals normally required for cell proliferation (3). Mostly through its transcriptional activity, p53 can inhibit cell cycle progression or induce apoptosis in response to stress or DNA damage. Moreover, p53 is known to be a critical factor in maintaining genomic stability and in eliciting DNA repair reactions (22, 32). Some earlier evidence also pointed to a regulatory role of p53 in transcriptionally activating a number of genes that could be involved in modulating cell movement (8-12). Two recent studies have begun to address the possibility that p53 might contribute to the regulation of cell movement and polarity (14, 15). By genetic deletion of the tumor suppressors, we show that p19Arf and p53, but not p27Kip1 or pRb, have a profound negative effect on cell motility and migration. p19Arf likely depends on p53 for migration regulation, and specific transcriptional activity, or specific target genes controlled by p53, may serve as the link between the nuclear events and the cytoskeleton, focal adhesion contacts, or extracellular matrix components to promote the migration phenotype. We are currently examining the candidate gene products whose expression profiles are altered by the p19Arf and p53 deficiencies in an effort to identify the molecule(s) responsible for the observed cellular effects.
The PI 3-kinase-Rac1 signaling module has been known to mediate growth
factor-stimulated cell motility (31). Activation of PI 3-kinase
(similar to deletion of the PTEN tumor suppressor, which is a
PIP3-specific phosphatase that counter-reacts with many PI
3-kinase effects) can cause activation of both Rac1 and Akt and
contributes to enhanced cell motility and invasion (20, 29). The cell
morphological and actin structural changes we have observed in
Arf/
and p53
/
MEFs, i.e. a round appearance and intense cortical actin
staining, are reminiscent of what was observed in the case of
PTEN
/
cells (20), suggesting that
elevation of the PIP3 level in the cells may be significant
to increased cell movement. Indeed, Rac1 and Akt appear to be important
contributors to the migration phenotype in PTEN deficiency (20, 29). A
few previous reports support the assumption that Rac may act
independently of Akt in signaling to mammalian cell migration (33, 34),
but a recent study employing constitutively active and dominant
negative Akt in fibroblasts shows that Akt is an essential mediator of
Rac-regulated cell motility stimulated through PI 3-kinase activation
(29). In the context of our finding that PI 3-kinase and Rac1 of this module are required for p19Arf- and p53-regulated
migration, it seems logical that a hierarchical link among them may be
at work to transduce signals resulting from the genetic defects of
these tumor suppressors to cell activities related to migration,
although the detailed relationship among them remains to be determined.
The components that serve to further relay signals from Rac1 and/or Akt
to cell motility might include PAK, phosphoinositide 5-kinase,
and WAVE-like actin polymerization molecules (31, 35).
Gadea et al. (15) have recently shown that p53 function is necessary for the suppression of Cdc42-induced filopodia formation and may transcriptionally control the initiating components of filopodia. Although conceptually our observation draws a similar conclusion regarding the negative effect of the Cdc42-mediated cell response imposed by p53, the mechanistic details of our cell motility results may differ from the interpretation of Gadea et al. (15), drawn mostly from the Cdc42 and/or p53 overexpression experiments. In the present work, although Rac1 and RhoA activities are shown to be up-regulated by deletion of Arf or p53, Rac1, but not RhoA nor Cdc42, appears to provide the key functional linkage between the p19Arf-p53 pathway and cell motility. However it remains likely that the contributions of Cdc42-mediated cell polarity and RhoA-mediated cell adhesion are an integral part of the p19Arf- and p53-regulated cytoskeleton organization and related functional output.
A recent gene array study has identified heparin-binding epidermal
growth factor-like growth factor (HB-EGF) as a p53-responsive gene
product that could be up-regulated by DNA damage and p53 induction
(36). HB-EGF may protect cells from
H2O2-induced apoptosis through MAPK
(mitogen-activated protein kinase) and PI 3-kinase activation,
presenting a possible autocrine regulatory loop linking p53 induction
with PI 3-kinase and Akt activation. Whether this p53-mediated
regulatory pathway is functional in the p53/
background remains unknown. In light of our finding that p53 deletion
would cause an up-regulation rather than a down-regulation of PI
3-kinase activity, it is likely that the above proposed p53 to HB-EGF
to PI 3-kinase pathway might be suppressed by another more dominant component unleashed by p53 deletion, which preferably
stimulates the PI 3-kinase-Rac1-Akt signaling module when the
p19Arf-p53 pathway is impaired. This possibility is
currently under investigation.
In summary, we have demonstrated that the p19Arf-p53 tumor
suppressor pathway negatively regulates cell motility, revealing an interesting connection between the established tumor suppressor pathway
and the PI 3-kinase-Rac1 signaling module that is known to modulate
cell migration. The results provide insights into a previously
under-appreciated cellular function of the tumor suppressor
pathway and may have important implications in developing strategies
that target Rho proteins in anti-cancer therapy.
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ACKNOWLEDGEMENTS |
---|
We thank members of Zheng laboratory for help with retroviral production and cDNA constructs and Dr. Martine Roussel for the gift of MEFs and Arf cDNA.
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FOOTNOTES |
---|
* This work was supported by grants from the National Institutes of Health (GM53943) and the Department of Defense Breast Cancer Program (BC990290) (both to Y. Z.).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.
To whom correspondence should be addressed. Tel.: 513-636-0595;
Fax: 513-636-3768; E-mail: yi.zheng@chmcc.org.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300341200
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ABBREVIATIONS |
---|
The abbreviations used are: MEF, mouse embryonic fibroblast; GST, glutathione S-transferase; PAK, p21-activated kinase; PI 3-kinase, phosphoinositide 3-kinase; GFP, green fluorescent protein; PIP3, inositol 1,4,5-trisphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; WASP, Wiskott-Aldrich syndrome protein.
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