1 Department of Biochemistry, Vanderbilt University School of Medicine,
Nashville, Tennessee, TN 37232-0146, USA
2 Department of Medicine, Vanderbilt University School of Medicine, Nashville,
Tennessee, TN 37232-0146, USA
* Author for correspondence (e-mail: graham.carpenter{at}mcmail.vanderbilt.edu )
Accepted 20 February 2002
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Summary |
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Key words: Phospholipase C, Anoikis, Insulin-like growth factor
![]() |
Introduction |
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PLC-1 and -
2 are tyrosine kinase substrates, and tyrosine
phosphorylation (Nishibe et al.,
1990
; Kim et al.,
1991
) is an essential step in their activation mechanism along
with membrane translocation (Todderud et
al., 1990
; Falasca et al.,
1998
). The
1 isoform is ubiquitously distributed in tissues
and cell lines, whereas the
2 isoform is predominantly expressed in
cells of the hematopoietic lineage (Homma
et al., 1989
; Rhee et al.,
1991
). Targeted gene disruption in mice shows that Plcg1
is essential for early embryonic development
(Ji et al., 1997
), whereas
mice nullizygous for Plcg2 are viable
(Wang et al., 2000
). Hence the
two
isoforms do not have redundant functions in the animal. A
Drosophila PLC-
has been identified and loss-of-function
mutations lead to aberrant wing vein development
(Thackeray et al., 1998
).
In the absence of pharmacological inhibitors, attempts to abrogate
PLC-1 function in growth-factor-stimulated cells have employed two
distinct approaches that have yielded discordant results. In one approach,
antibodies to PLC-
1 or fragments of PLC-
1, acting as
dominant-negative molecules, have been microinjected into quiescent cells
prior to treatment of the cells with platelet-derived growth factor (PDGF) or
epidermal growth factor (EGF) (Roche et
al., 1996
; Wang et al.,
1998
). The conclusion drawn from these experiments is that
PLC-
1 is essential for mitogenesis induced by these growth factors. In
separate experiments using different cell lines, mutagenesis of the
PLC-
1 specific association site on the PDGF ß-receptor has been
employed to prevent PLC-
1 activation in PDGF-treated cells. The
conclusion of these experiments is that the loss of PLC-
1 activation
has either no effect (Ronnstrand et al.,
1992
; Rosenkranz et al.,
1999
) or produces a small (30%) decrease in mitogenesis
(Valius et al., 1993
).
We have used mouse embryos from gene disruption experiment to establish
immortalized cell lines of defined Plcg1 genotypes
(Ji et al., 1997). The
Plcg1 nullizygous cells do not mobilize Ca2+ in response
to growth factors, whereas wild-type cells
(Ji et al., 1997
) or Null
cells in which PLC-
1 has been re-expressed
(Ji et al., 1999
) do
demonstrate a Ca2+ response to growth factors. Comparison of these
cell lines in mitogenesis assays has not shown a requirement for PLC-
1
in the induction of DNA synthesis by EGF
(Ji et al., 1998
) or PDGF
(Liao et al., 2001
).
The control of fibroblastic cell proliferation involves not only signaling
from receptor tyrosine kinases but also signaling from adhesion receptors
(Schwartz, 1997;
Giancotti and Ruoslahti, 1999
;
Aplin et al., 1999
). Integrin
activation is known to increase intracellular Ca2+, although it
remains unclear whether this involves PLC-
or another mechanism
(Clark and Brugge, 1995
;
Sjaastad and Nelson, 1996
). It
is possible, therefore, that adhesion-dependent signals are able to substitute
for the loss of PLC-
1 from growth factor receptor signaling pathways.
Therefore, we have attempted to determine whether PLC-
1 has a role in
the survival of cells placed in suspension and in the absence of
adhesion-dependent signaling.
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Materials and Methods |
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Cell line
Spontaneously immortalized Plcg1-/- mouse embryonic
fibroblasts, referred to as Null cells, were prepared from E9.5 mouse embryos
with targeted disruption of the Plcg1 gene
(Ji et al., 1997). The rat
PLC-
1 full-length cDNA (a generous gift from Sue Goo Rhee, National
Institutes of Health) was stably expressed in the Plcg1-/-
cells by retroviral infection to generate PLC-
1 re-expressing cells
referred to as Null+ cells (Ji et al.,
1999
). Null+ cells have been derived from two independent Null
cell lines referred to as targeting vector (TV)-I and TV-II. Unless otherwise
stated, TV-I Null and Null+ cells are employed in the experiments reported in
this manuscript. All cells were cultured in Dulbecco's Modified Eagle's Medium
(DMEM) containing 10% fetal bovine serum (FBS) and were incubated at 37°C
in a humidified atmosphere with 5% CO2.
Suspension culture
Regular cell culture dishes were coated with a film of poly-HEMA following
the protocol reported by Folkman and Moscona
(Folkman and Moscona, 1978).
Briefly, a solution of 120 mg/ml of polyHEMA in 95% ethanol was mixed
overnight, centrifuged at 800 g to remove undissolved particles and
diluted 1:10 in 95% ethanol. The resulting solution was then placed in the
culture dishes. The dishes were left to dry at room temperature overnight.
Before use, the coated dishes were washed twice with phosphate buffered saline
(PBS).
Anoikis protocol
Near-confluent cells on regular plastic tissue culture dishes were
trypsinized to produce a single cell suspension. Trypsin was inactivated by
the addition of either soybean trypsin inhibitor or 10% FBS. Approximately
2.5x105 cells were placed in each well of a sixwell dish
precoated with poly-HEMA to prevent cell attachment. At the indicated times,
cells were collected, washed with PBS and resuspended by trypsinization.
Viable cells were counted in a hemocytometer using the trypan blue exclusion
procedure.
Caspase 3 activity assay
Caspase 3 activity present in cells under different culture conditions was
measured using a fluorometric assay (Pharmingen Inc.) according to the
manufacturer's protocol. In brief, cells were harvested, washed with PBS and
lysed by incubating with chilled cell lysis buffer for 15 minutes at 4°C.
The supernatant was collected by centrifugation at 14,000 g for 15
minutes, and the protein concentration was measured by the Bradford method
using bovine serum albumin as the standard. Cell extracts (30 µg protein)
were incubated for 1 hour at 37°C in a protease assay buffer containing
the caspase-3-specific substrate N-acetyl-Asp-Glu-Val-Asp-AMC
(7-amino-4-methylcoumarin). After 1 hour of incubation, the release of the
fluorogenic compound AMC from the substrate was measured in a
spectrofluorometer at an excitation wavelength of 380 nm and an emission
wavelength at 440 nm. The fluorescence unit (2.5x106) for the
emission at 440 nm for PLC-1 Null cell in serum-free condition was
considered as 100%.
Immunoprecipitation and western blotting
Cells were placed in 100 mm tissue culture dishes and incubated at 37°C
for 48 hours in medium containing 10% FBS. At a confluence level of 50-70%,
the cells were serum-starved by incubating overnight in medium containing 0.5%
FBS. After trypsinization, the cells were placed in suspension culture in
poly-HEMA-coated dishes. IGF-I (100 ng/ml) was added as indicated, and the
cells were incubated at 37°C for 15 minutes. The cells were then
collected, washed with PBS and lysed by incubating with TGH lysis buffer for
20 minutes at 4°C. Insoluble materials were then removed by centrifugation
(14,000 g for 10 minutes) at 4°C. Protein concentrations were
assayed by the method of Bradford. To immunoprecipitate the IGF-I receptor or
PLC-1, the primary antibodies were incubated with 1 mg of cell lysate
overnight at 4°C. This was followed by a 1 hour incubation with Protein A
Sepharose. Immune complexes were subsequently washed three times with TGH
lysis buffer, resuspended in 1xLaemmli buffer and boiled for 5 minutes.
The samples were then electrophoresed on an 8% SDS-polyacrylamide gel and
subsequently transferred to nitrocellulose membranes for western blotting.
Blotting was performed as described elsewhere
(Ji et al., 1999
). Bound
antibody was detected by enhanced ECL.
![]() |
Results |
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To explore the role of PLC-1 in cell survival signaling, we compared
the sensitivity to cell death of Plcg1 Null and Null+ cells in the
absence of cell adhesion. Trypsinized cells were plated in tissue culture
dishes or in dishes pre-coated with poly-HEMA to prevent cell attachment. Cell
viability was determined in these adherent and suspension cultures in the
presence or absence of 10% FBS as a source of growth-factor-dependent survival
signals. Trypan blue staining was employed to calculate the percentage of
non-viable cells 20 hours after plating. The data in
Fig. 1A show that, when Null
and Null+ cells are placed in suspension under serum-free conditions, cell
death occurs at nearly equivalent levels (approximately 60%) in both cell
types. In the presence of serum, however, cell death was considerably higher
in the Null cells (35%) compared with the Null+ cells (5%). When the Null and
Null+ cells are allowed to adhere to the culture dish surface in the presence
or absence of serum for the same amount of time, the data show that no
significant cell death is induced in either Null and Null+ cells.
|
These results indicate that Null+ cells demonstrate the expected characteristics of fibroblasts for cell survival in suspension. Namely, that the presence of serum growth factors prevent cell death. However, Null cells in suspension are significantly less protected from cell death by serum growth factors. Similar results were obtained when cell death was assayed by propidium iodide exclusion.
Cell death measured under these conditions for Null and Null+ cells can be
considered to reflect programmed cell death because of the DNA fragmentation
or laddering (data not shown) and the induction of caspase 3 as shown in
Fig. 1B. In this experiment,
caspase 3 was induced to about the same level in Null and Null+ cells placed
in suspension under serum-free conditions. However, in suspension cultures to
which serum was added to promote survival, caspase induction in Null cells was
three-fold greater than in Null+ cells. Therefore, the presence of
PLC-1 is likely to be necessary molecule for serum-mediated survival of
cells deprived of matrix attachment. The addition of the caspase inhibitor
Z-VAD-FMK confirms that the proteolytic activity measured in this assay is
caused by caspase activity.
Influence of IGF-I on the survival of Null and Null+ cells
IGF-I is a growth factor component of serum and is considered to be a major
inhibitor of apoptosis in serum (Baserga et
al., 1997). Therefore, we examined the influence of IGF-I on the
survival of Null and Null+ cells placed in suspension under serum-free
conditions. In addition, we tested a second independent pair of Null and Null+
cells, referred to as TV-II, for their capacity to survive under suspension
conditions. In Fig. 2A,B, the
results of cell viability assays (trypan blue staining) are reported, and the
induction of caspase 3 activity is shown in
Fig. 2C,D. Both assays show
that IGF-I protects Null+ cells much more effectively than Null cells from
cell death induced by suspension culture. Also, comparable results in both
assays were obtained for TV-I Null and Null+ cells
(Fig. 2A,C) and the
independently derived TV-II Null and Null+ cells
(Fig. 2B,D). These cell lines
are also responsive to mitogenic growth factors, such as EGF and PDGF, which
may also act as survival factors. When each of these growth factors was tested
in the anoikis protocol, they promoted only a low level of survival
(approximately 20%) compared with IGF-I.
|
Since IGF-I-mediated cell survival signaling in suspension culture is impaired in the Null cells, the IGF-I receptor level and IGF-I induced receptor phosphorylation in Null and Null+ cells were compared. The results are depicted in Fig. 3 and demonstrate that Null and Null+ cell lines have comparable levels of IGF-I receptors and that the receptors are autophosphorylated in a ligand-dependent manner in both cell lines.
|
PLC-1 function in suspension culture
The data presented in this paper suggest that PLC-1 is necessary for
IGF-I-mediated cell survival in suspension culture. If this is a critical
difference between Null and Null+ cells in these assays, then
12-O-tetradecanoyl phorbol-13-acetate (TPA) and ionomycin, pharmacological
agents that replace downstream PLC-
1 functions, should promote survival
of cells that do not contain PLC-
1. Ionomycin raises intracellular
Ca2+ levels, whereas TPA constitutively activates protein kinase C.
To test this possibility, Null cells were placed in suspension and treated
with IGF-I or ionomycin and TPA or IGF-I together with ionomycin and TPA.
Subsequently, caspase 3 activity was measured as an index of cell death. The
results are shown in Fig. 4. As
expected from previous data, the presence of IGF-I did not significantly lower
caspase 3 activation when the Null cells were placed in suspension. Treatment
with ionomycin and TPA, however, decreased caspase 3 activation by almost 40%,
suggesting that pharmacological replacement of PLC-
1 function does
enhance cell survival. To test whether IGF-I signaling might further increase
cell survival in the presence of TPA and ionomycin, all three agonists were
added to the suspension of Null cells. Under these conditions, caspase 3
activation was reduced by approximately 70%. The results of this experiment
are consistent with the conclusion that PLC-
1 activity contributes to
IGF-I-induced cell survival pathways. The increased level of reduction of
caspase 3 activity by ionomycin and TPA in the presence of IGF-I indicates
that IGF-I contributes signals to cell survival that are not entirely replaced
by ionomycin and TPA.
|
If PLC-1 is involved in IGF-I-dependent survival of cells placed in
suspension, then it would be expected that IGF-I might induce the tyrosine
phosphorylation of PLC-
1 under these conditions. This has been tested
in the experiment shown in Fig.
5 using Null+ that are adherent or placed in suspension. In either
situation, the addition of IGF-I did not provoke a detectable level of
PLC-
1 phosphorylation. However, in the presence of the phosphotyrosine
phosphatase inhibitor pervanadate and IGF-I the tyrosine phosphorylation of
PLC-
1 was readily detected. Pervanadate by itself did not yield
detectable phosphorylation of this protein.
|
When cells are placed in suspension culture, recent evidence indicates that
the cellular content of some signaling proteins, for example, Ras
(Gatzka et al., 2000) and
insulin receptor substrate-1 (IRS-I)
(Leburn et al., 2000
) are
dramatically decreased within a few hours. Also, it has been reported that the
induction of apoptosis by etoposide in Molt-4 cells results in the
caspase-dependent cleavage of PLC-
1
(Bae et al., 2000
). In view of
these reports we have examined the metabolic stability of PLC-
1 in
Null+ cells placed in suspension. The results showed that during a 10 hour
incubation in suspension culture, in serum-free medium without IGF-I, the
cellular level of PLC-
1 was unaltered (data not shown). Therefore,
these results do not suggest that PLC-
1 is metabolically destabilized
by suspension culture.
Influence of PI-3 kinase and MAP kinase pathways on IGF-I survival
signaling
The two major cell signaling pathways utilized by IGF-I in protection from
apoptosis are the PI-3 kinase and mitogen-activated protein (MAP) kinase
pathways. In view of the potential role of PLC-1 signaling in
IGF-I-dependent cell survival, the capacity of pharmacologic inhibitors of
PI-3 kinase and MAP kinase activation were tested for their influence on
IGF-I-mediated survival of Null+ cells in suspension culture. Null+ cells were
treated with LY294002, a stable inhibitor of PI-3 kinase, and/or PD98059, an
inhibitor of MAP kinase activation, prior to the addition of IGF-I. Caspase 3
activity was then measured 20 hours after plating. The results, presented in
Fig. 6, show that in the
presence of LY294002 or PD98059, the capacity of IGF-I to prevent caspase 3
activation was reduced by approximately 50%. However, when the two inhibitors
were added together, the protective effect of IGF-I on caspase 3 induction was
completely prevented. This suggests that the IGF-I-dependent survival of these
cells in suspension is also dependent on signaling through PI-3 kinase and MAP
kinase.
|
![]() |
Discussion |
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The major signaling pathways activated by ligand occupation of the IGF-I
receptor are the Ras/MAP kinase pathway and the PI-3 kinase-dependent
activation of Akt, and both of these pathways are reported to be necessary for
the anti-apoptotic function of IGF-I
(Kulik et al., 1997; Kulik et
al., 1998; Parrizas et al.,
1997
; Peruzzi et al.,
1999
). This is consistent with our data showing that inhibitors of
these pathways prevent IGF-I from protecting Null+ cells from cell death in
suspension culture. Recently, a third IGF-I signaling pathway that
participates in cell survival has been identified in 32 D cells
(Peruzzi et al., 1999
). This
pathway depends on raf activation and translocation to mitochondria. All three
of these pathways are known to promote the phosphorylation of BAD and thereby,
at least in part, prevent apoptosis.
The means by which PLC-1 may participate in antiapoptotic signaling
is unclear. Ca2+ can induce or prevent apoptosis depending on the
cell line and the nature of the calcium signal
(Berridge et al., 1998
). IGF-I
has been shown to promote Ca2+ entry into the cells, inositol
1,4,5-trisphosphate formation, the production of 1,2-diacylglycerol and
activation of protein kinase C (Kojima et
al., 1988
; Kojima et al.,
1990
; Kojima et al.,
1993
; Takasu et al.,
1989
; Munaron and Pla,
2000
). These intracellular signaling events are consistent with
the activation of a PLC isoenzyme. The tyrosine phosphorylation of
PLC-
1 induced by IGF-I, which is constitutively activate IGF-I
receptors, or insulin has been reported for adherent cells
(Jiang et al., 1996
;
Foncea et al., 1997
;
Hong et al., 2001
; Eichhorn et
al., 2001), and our data extends this to IGF-I-treated cells in
suspension.
In our study the addition of pervanadate was necessary to detect
PLC-1 tyrosine phosphorylation. This seemingly low level of
phosphorylation may be influenced by two factors. First, in all cell systems
the agonist-dependent tyrosine phosphorylation of PLC-
1 is transient,
and the detected level of tyrosine phosphorylation therefore depends on the
level of synchrony in the cell population response to the agonist. Second,
others have shown that phosphotyrosine phosphatase activity is increased when
cells are placed in suspension (Sabe et
al., 1997
). Under these conditions the addition of pervanadate is
effective in revealing the presence of tyrosine phosphorylated proteins.
However, in our experiment pervanadate was also required to detect
PLC-
1 tyrosine phosphorylation in adherent cells treated with IGF-I.
This does suggest that IGF-I is not a particularly strong stimulator of
PLC-
1 tyrosine phosphorylation. However, it is possible that the
observed level of tyrosine phosphorylation is sufficient to transiently
activate PLC-
1 or that pervanadate only partially overcomes phosphatase
activity and in situ PLC-
1 is actually phosphorylated to a higher
extent. Also, several mechanisms have been proposed to enhance the activation
of PLC-
1 in concert with tyrosine phosphorylation or in its absence
(Rhee, 2001
). These include
potential modulatory molecules such as phosphatidic acid, phosphatidylinositol
3,4,5-trisphosphate, arachidonic acid and the 680k Da protein AHNAK. Whether
any of these are relevant to the IGF-dependent control of PLC-
1
activity during anoikis is not known.
A mechanism for IGF-I-induced Ca2+ influx in cells is reported
to involve the IGF-I-dependent translocation of a calcium-permeable channel to
the plasma membrane through a PI-3-kinase-dependent signal
(Kanzaki et al., 1999). The
means by which the opening of this channel is regulated at the cell surface is
not known. However, this channel is structurally related to the
transient-receptor-potential (TRP) channel family, and these channels are
thought to be activated by the depletion of intracellular stores of calcium by
an unclear mechanism (Putney,
1999
). Hence, PLC-
1 activation by IGF-I could provide a
means to empty intracellular calcium stores and thereby provoke influx of
extracellular calcium through this TRP channel.
Interestingly, cell adhesion is known to regulate the plasma membrane level
of PI 4,5-P2. When cells are placed in suspension culture, cellular
PI 4,5-P2 levels decline significantly and are restored quickly
when cells are allowed to adhere to a substratum
(McNamee et al., 1993;
Oude Weernink et al., 2000
).
This change is thought to reflect changes in the activity of PI4P-5 kinase.
However, suspension also increases the cellular level of inositol phosphates
provoked by agonists, such as PDGF, that act on PLC-
1. Hence, the
reductions in PI 4,5-P2 levels provoked by suspension conditions do
not seem to sufficiently abrogate PLC-
1 activation through the receptor
tyrosine kinases.
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Acknowledgments |
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