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(Received for publication, October
16, 1995) From the
Phosphatidic acid (PA) is a putative novel messenger in signal
transduction and membrane traffic. We have synthesized a photolyzable
derivative of PA, termed caged PA (cPA), which may be utilized as a new
tool in studies of PA-mediated cellular events.
1-(2-Nitrophenyl)diazoethane, synthesized from 2-nitroacetophenone, was
reacted with dipalmitoyl-PA to yield a 1-(2-nitrophenyl)ethyl ester of
PA. Photolysis of the compound by ultraviolet light resulted in the
formation of phosphatidic acid. The structure of the compound and of
its photolytic products was verified by NMR spectroscopy. The utility
of cPA was examined in HT 1080 metastatic fibrosarcoma cells, in which
the formation of PA by phospholipase D was implicated in
laminin-induced release of gelatinase A (matrix metalloproteinase 2
(MMP-2)). The uptake of cPA by HT 1080 cells reached a plateau after
120 min of incubation. Ultraviolet illumination of cPA-loaded cells for
5 s resulted in photolysis of 1.8% of the cell-incorporated cPA. The
photolysis of cPA caused a 2-fold elevation in the release of MMP-2 to
the medium, whereas nonphotolyzed cPA caused no change in MMP-2
release. Moreover, the effect of cPA photolysis was significantly
higher than that obtained with extracellularly introduced PA. Thus, the
effect of laminin on MMP-2 secretion can be mimicked by photolysis of
cPA, suggesting a pivotal role for phospholipase D in laminin-induced
cancer cell invasiveness and metastasis. These results indicate that
cPA could serve as a unique tool for studying the cellular roles of PA.
Phospholipase D (PLD) ( Here we describe the synthesis and
characterization of a photolyzable analogue of PA, termed caged PA
(cPA), that allows the elevation of the intracellular level of PA upon
ultraviolet irradiation. The utility of cPA as a probe was examined in
HT 1080 metastatic fibrosarcoma cells. Metastatic spread depends upon
the invasive capacity of tumor cells which, in turn, depends on their
ability to secrete proteolytic enzymes, such as gelatinase A (MMP-2),
that participate in degradation of the basement
membrane(6, 7, 8) . Laminin, an important
component of the basal membrane, stimulates MMP-2
secretion(9, 10, 11) . We have shown recently
that extracellularly introduced laminin activates PLD in HT 1080
cells(12) . Laminin-induced release of MMP-2 was inhibited by
1-butanol, while addition of exogenous PA or of bacterial PLD into the
growth medium mimicked the effect of laminin(12) . Thus, HT
1080 cells represent a useful experimental system for studying the
second messenger function of PA. We now show that cPA photolysis leads
to a rise in intracellular PA and subsequently to secretion of MMP-2 in
HT 1080 cells.
The synthesis of cPA is based on alkylation of the weakly
ionized phosphate group of PA by 1-(2-nitrophenyl)diazoethane (Fig. 1A). In the first step,
1-(2-nitrophenyl)hydrazonoethane is synthesized from
2-nitroacetophenone by refluxing it with hydrazine hydrate in the
presence of protons donated by glacial acetic acid. In the second step,
1-(2-nitrophenyl)hydrazonoethane is oxidized by MnO
Figure 1:
Synthesis (A)
and photolysis (B) of cPA. A, in the first step of
cPA synthesis, 2-nitroacetophenone was reacted with hydrazine hydrate
to yield 1-(2-nitrophenyl)hydrazonoethane. Activation of this compound
with MnO
The
expected structure of cPA and its photolytic products was verified by
comparing the proton NMR spectra of cPA, PA, and photolyzed cPA (Fig. 2). The spectrum of cPA clearly shows the additional peaks
contributed by the alkylating group (Fig. 2A). In
particular, note the aromatic signals of the nitrophenyl group and the
distinct appearance of the doublet at 1.7 ppm and the quartet at 3.0
ppm due to the coupled CH
Figure 2:
Proton NMR spectrum of cPA and its
photolytic products. The proton NMR spectrum of cPA was compared to
that of PA (A) and of photolyzed cPA (B). The doublet
in the cPA spectrum at 1.7 ppm is due to the ethyl ester CH
Next we have measured the incorporation of cPA into HT 1080
cells by introducing [
Figure 3:
Uptake (A) and photolysis (B) of cPA in HT 1080 cells. A, serum-deprived cells were incubated with 16 µM
[
MMP-2 is a key determinant of the metastatic potential
of tumor cells. MMP-2 production in HT 1080 is stimulated by
laminin(9, 10, 11) . We have previously
demonstrated that in HT 1080 cells laminin activates PLD (12) .
We hypothesized that the rise in PA level elicits MMP-2 release and
that cPA photolysis would mimic this effect. The effect of cPA
photolysis on MMP-2 release was compared to that of exogenously added
PA (Fig. 4). cPA had no effect on MMP-2 release in
nonilluminated cells (B) or in cells illuminated before its
addition (A). Photolysis of cPA in cPA-loaded cells (C) caused a 2.1-fold increase in the activity of MMP-2
measured in the growth medium. In comparison, exogenous PA elevated
MMP-2 release by 66% in illuminated cells. Incubation of HT 1080 cells
with the by-product of cPA photolysis, 2-nitrosoacetophenone, had no
effect on MMP-2 release, even when incubated with the cells for 3 h at
very high concentrations (100 µM) (data not shown). It may
thus be concluded that cPA photolysis mimics the effect of laminin in
these cells by elevating the level of PA.
Figure 4:
Induction of MMP-2 release upon
illumination of cPA-loaded cells. DMEM-PR, 0.1% BSA (bars
1, 3, 4, and 6) or DMEM-PR, 0.1% BSA
containing 16 µM cPA (bars 2, 5, and 7) were added to nonilluminated cells (B and C) or to 5-s illuminated cells (A). After 1 h, the
medium was removed and all plates were washed twice with DMEM-PR, 0.1%
BSA. cPA photolysis was accomplished by ultraviolet illumination for 5
s (C, bar 7) while control cells were not illuminated (bars 2 and 5). PA was added to a final concentration
of 50 µg/ml (bars 3 and 6). After a 2
The optimal duration of
irradiation was tested by illuminating cPA-loaded cells for various
times and measuring release of MMP-2 (Table 1). It is illustrated
that short photolysis was more effective than long photolysis. As
demonstrated earlier (Fig. 1B), efficient in vitro photolysis of cPA required irradiation longer than 60 s. However,
exposing cells to ultraviolet irradiation for more than 20 s
dramatically reduced their viability.
To identify cellular processes that are directly modulated by
PA, it is necessary to be able to experimentally cause very rapid
changes in cellular PA concentrations. The introduction of caged PA
derivatives into cultured cells is a novel approach that allows
experimental elevation of intracellular PA. Light-induced generation of
PA mimics signal-dependent activation of PLD, while physiological
receptor, transducer, and effector mechanisms are bypassed. This
approach offers the possibility of eliciting very rapid changes in
membrane levels of PA in the absence of parallel, hormone-induced
activation of other signaling pathways. In the present study it was
demonstrated that cPA photolysis stimulates MMP-2 release whereas
nonphotolyzed cPA had no effect, and that the effect of photolyzed cPA
is about twice the effect of extracellularly added PA. The effect of
cPA photolysis is likely due to the elevation of intracellular PA.
Illumination for 5 s caused photolysis of 1.8% of the cPA (24 pmol/5
The utilization of cPA offers a number of possibilities for
studying the metabolism and action of PA in cells and cell-free
preparations. Caged PA derivatives with either radioisotopically
labeled or fluorescently labeled phosphatidyl moieties of different
fatty acyl chain length could be employed to follow the uptake,
transport, and metabolism of cPA and PA, before and after photolysis,
respectively. This could provide hitherto unobtainable information
regarding the metabolic fate of the PA produced by the signal-activated
PLD. In addition, cPA photolysis could potentially be exploited to
identify immediate biochemical responses that are directly regulated by
PA, e.g. changes in activity of specific enzymes, protein
phosphorylation patterns, or ionic currents. At present, the utility
of cPA is somewhat limited by the low efficiency of the loading step.
It may be expected (23, 24) that the exchange of cPA
with cells (and hence the efficiency of its uptake) will be improved by
using short chain analogs of cPA. Another factor that limits the
usefulness of cPA is its apparent toxicity in some cell lines. The
mechanism of this effect is not clear. Certain cell types are more
sensitive to the ``cage'' moiety utilized in
cPA(15, 16) . One way to overcome this problem will be
to use photosensitive blocking groups other than the
1-(2-nitrophenyl)ethyl moiety that was employed here. We have
previously shown that laminin stimulates PLD activity in HT 1080
cells(12) . A causal relationship between PLD activation and
the subsequent laminin-induced release of MMP-2 was suggested by the
fact that the effect of laminin was inhibited by 1-butanol, an
alternative substrate of PLD that attenuates PA production by shunting
phosphatidyl moieties from PA into phosphatidylbutanol. Furthermore,
laminin-induced release of MMP-2 could be mimicked by treatment of the
cells with an exogenous (bacterial) PLD. In the present study it is
shown that light-induced generation of PA by photolysis of cPA also can
mimic the action of laminin on MMP-2. Collectively, these data strongly
suggest a pivotal role for PLD and PA in the signaling cascade of
laminin-induced MMP-2 release and, consequently, in tumor cell
invasiveness and metastasis. The photolysis of cPA is likely to be
useful in elucidating the downstream biochemical events that follow PLD
activation in these cells, causing changes in gene expression and
culminating in malignant dissemination.
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 29656-29659
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)was shown to be activated by
agonists such as platelet-derived growth factor, epidermal growth
factor, the chemotactic peptide formyl-Met-Leu-Phe, vasopressin,
gonadotropin-releasing hormone, bombesin, and many
others(1, 2, 3) . It is assumed that upon
activation of PLD a signal is transmitted downstream via the rise in
intracellular phosphatidic acid (PA). When introduced extracellularly,
PA was shown to mimic some of the effects of these
agonists(4, 5) . The mechanism(s) by which PA affects
cell function and the identity of its intracellular targets are still
not known. Moreover, due to the lack of specific activators or
inhibitors of PLD, the causal relationship between PLD-mediated
elevation of cellular PA and any specific cell response has not been
demonstrated with certainty.
Materials
All chemicals and solvents used were
of reagent grade. Dipalmitoylphosphatidic acid (sodium salt),
imidazole, and bovine serum albumin (BSA) were obtained from Sigma.
Gelatin was a product of BDH. Hydrazine hydrate, CDCl
, and
2-nitroacetophenone were from Aldrich. MnO
(activated
grade), MgSO
, and Silica Gel 60 aluminum thin layer
chromatography (TLC) plates were purchased from Merck.
[P]ATP and [
C]PA were
obtained from DuPont NEN. Diacylglycerol kinase was purchased from
Calbiochem. Dulbecco's modified Eagle's medium (DMEM),
fetal calf serum (FCS), and supplemented antibiotics are products of
Biological Industries, Kibbutz Beth HaEmek, Israel.Cell Lines and Tissue Culture
HT 1080 cells
derived from a metastatic lesion of human fibrosarcoma were obtained
from the American Type Culture Collection (ATCC) and maintained in
DMEM, 10% FCS supplemented with 1000 units/ml penicillin and 100 mg/ml
streptomycin. The experiments were carried out with cells which have
been serum-deprived by an overnight incubation in DMEM, 0.1% BSA.
Before the experiment, the medium was replaced by DMEM without phenol
red (DMEM-PR) containing 0.1% BSA to avoid quenching of ultraviolet
light by the dye.Preparation of the Free Acid Form of
PA
Phosphatidic acid (sodium salt) was dissolved in chloroform
to yield a final concentration of 14 mg/ml. Methanol and 0.2 N HCl were added (chloroform/methanol/HCl ratio of 1:1:0.9, v/v),
and the mixture was mixed vigorously. Phase separation was accomplished
by centrifugation for 10 min at 2000 rpm (Sorval HS-4) at room
temperature. The lower phase containing the free acid form of PA was
collected, and the pH was adjusted to 4-5 with 10% triethylamine
in chloroform.Synthesis of cPA
The preparation of
1-(2-nitrophenyl)hydrazonoethane was carried out by dissolving 0.826 g
(5 mmol) of 2-nitroacetophenone, 0.562 g (11.2 mmol) of hydrazine
hydrate, and 0.32 ml (5 mmol) of glacial acetic acid in 10 ml of
ethanol in a 20-ml reflux receptacle. The mixture was refluxed while
stirring for 3 h and then was allowed to cool. The ethanol was
evaporated in a 25-ml round-bottom flask using a rotary evaporator, and
the residue was dissolved in 2 ml of chloroform. Traces of acetic acid
were removed by washing the chloroform three times with 1 ml of
HO. Anhydrous MgSO (0.5 g) was added, and the
mixture was left at room temperature for 5 min, then filtered through
cotton, and stored at -20 °C. For the synthesis of
1-(2-nitrophenyl)diazoethane, 1.35 ml of chloroform were stirred
vigorously with 0.15 ml of 1-(2-nitrophenyl)hydrazonoethane together
with 255 mg of MnO. After 5 min, the mixture was filtered
through cotton. This stage and all following stages were performed in
subdued light. 1-(2-Nitrophenyl)diazoethane was freshly prepared before
each synthesis of cPA and used immediately. The final stage in the
preparation of 1-(2-nitrophenyl)ethyl ester of PA (namely cPA) was
accomplished by stirring PA-free acid solution (10.35 µmol)
together with the newly prepared 1-(2-nitrophenyl)diazoethane (155.25
µmol) for 45 min. Then the reaction mixture was applied to an
aluminum TLC plate and developed with chloroform, methanol, 25%
ammonium hydroxide (65:25:5, v/v). The band of 1-(2-nitrophenyl)ethyl
ester of PA was localized by its fluorescence upon illumination of the
edges of the plate at a wavelength of 254 nm. The band (R
about 0.56) was scraped, and the cPA was
extracted with methanol and quantitated by a phosphate assay as
described before(13) .Preparation of
PA labeled with P- and
C-Labeled
cPAP was prepared from
diacylglycerol to which
P was transferred from
[
-
P]ATP by diacylglycerol kinase as
described previously(14) . Mixing the
P-labeled or
C-labeled PA with PA dissolved in chloroform was followed
by desalting as described above. [P]cPA and
[
C]cPA were prepared as detailed above for
nonlabeled cPA.NMR Spectroscopy
NMR spectra were obtained on a
Bruker AMX 400 NMR spectrometer. The sample was dissolved in CDCl to a final concentration of 2 mg/ml. Peak assignment was aided by
comparison of the precursors and the cPA. Data were analyzed on a
Personal Iris Workstation (Silicon Graphics).Loading of Cells with cPA and Its Photolysis
cPA
was solubilized by a 30-min sonication in a bath sonicator in DMEM-PR,
0.1% BSA. The solubilized cPA was introduced into the medium of
overnight serum-deprived HT 1080 cells in a final concentration of 16
µM for 1 h, unless otherwise indicated. At the end of the
incubation, excess cPA was removed by washing the cells twice with
DMEM-PR, 0.1% BSA. Photolysis was carried out by illuminating the
culture plates from a distance of 10 cm with two 15-watt mercury lamps
(315 and 365 nm wavelength).Determination of Cellular PA Mass
Lipids were
extracted from cultures of resting HT 1080 cells (5) and
separated by two-dimensional TLC using
chloroform/methanol/NHOH (65:25:5) in the first dimension
and chloroform/acetone/methanol/acetic acid/water (30:40:10:10:5) in
the second dimension. Standard curves of PA were run in parallel. PA
mass was determined by Coomassie Blue staining essentially according to
Kaszkin et al.(19) .Determination of MMP-2 Activity
MMP-2 activity was
determined as described previously(12) . Results are expressed
as the measured intensity relative to that of control.
to
yield 1-(2-nitrophenyl)diazoethane which later was allowed to react
with PA resulting in the desired 1-(2-nitrophenyl)ethyl ester of PA. In
general, the preparation of cPA is similar to the preparation of other
caged compounds(15, 16, 17) . The main
difference is in the last stage of the preparation which was
accomplished in a single phase since here both reactants are
hydrophobic and soluble in chloroform. As demonstrated in Fig. 1B, the 1-(2-nitrophenyl)ethyl moiety, which in
cPA is linked to the phosphate group of the PA, is removable upon
ultraviolet illumination at a wavelength of 300-400 nm.
has led to 1-(2-nitrophenyl)diazoethane, which is
able to react with desalted PA to yield the desired cPA. NPE,
1-(2-nitrophenyl)ethyl. B, the 1-(2-nitrophenyl)ethyl moiety
can be removed by ultraviolet illumination yielding PA and
2-nitrosoacetophenone. [C]cPA was loaded on a
TLC plate and then ultraviolet illuminated for 1 min (right
lane). A nonphotolyzed sample of [C]cPA was
added (left lane), and the plate was developed with
CHCl, MeOH, 25% ammonia (65:25:5, v/v), followed by
autoradiography.
-CH of the ethyl ester
(J
group split by J-coupling (7.2 Hz) with the adjacent CH group
(quartet at 3.0 ppm) of the phosphoester. The signals labeled CH and CH are assigned to the lipid chains of
PA.
P]cPA into the growth
medium. The uptake of cPA was time-dependent, reaching a plateau after
120 min (Fig. 3A). In all additional experiments,
loading was accomplished by incubating the cells with cPA for 1 h,
after which uptake was 1327 pmol/5
10
cells,
representing 8.3% of the cPA introduced into the medium. In vivo photolysis was demonstrated in
[P]cPA-loaded cells that were irradiated for
various times (Fig. 3B). There was a direct
relationship between the time of irradiation and
[
P]PA accumulation. The results demonstrate that
cPA is incorporated into the cells in a time-dependent manner. It is
also shown that the increase in PA is correlated to the length of
irradiation. The uptake of cPA was studied also in NIH 3T3 and Swiss
3T3 cells. These cells were found to be much more sensitive to an
apparently cytotoxic effect of cPA at the concentrations used. (
)
P]cPA for the indicated time. Following
incubation and an extensive wash, the lipids were extracted
and the incorporation was determined by measuring the radioactivity in
the samples. Each data point represents the mean ± S.D. of three
determinations. B, cells were loaded with cPA by incubation
for 1 h in DMEM, 0.1% BSA, 16 µM
P-labeled cPA. Following incubation and an extensive
wash, the cells were irradiated for the indicated time as
described under ``Experimental Procedures.'' The lipids were
extracted, dried, and separated by TLC with chloroform, methanol, 25% NH
OH (65:25:5, respectively) as the mobile phase. The autoradiogram of the plate
was densitometrically analyzed in order to calculate the percentage of
photolysis.
-h
incubation at 37 °C, 75 µl of medium samples were removed and
MMP-2 activity was tested as described under ``Experimental
Procedures.'' The results are expressed as the activity relative
to control (no treatment, bar 4).
On the other hand, as
demonstrated here, a 5-s illumination was sufficient to photolyze
enough cPA to mimic the effect of laminin on MMP-2 secretion. The
reduction in the effectiveness of cPA photolysis on MMP-2 release,
found with illumination periods longer than 5 s, might be explained by
the greater damage caused to the cells by the ultraviolet light.
10 cells). The mass of PA in resting HT 1080 cells
was determined by a two-dimensional TLC/Coomassie Blue staining
procedure and found to be 31 pmol/5 10 cells.
Therefore, it seems that even a relatively small elevation in PA mass
is sufficient for eliciting a significant cellular response. In other
studies it has been shown that agonist activation caused elevations of
PA mass that were higher by approximately one order of
magnitude(18, 19, 20, 21, 22) .
These greater elevations were obtained with a high concentration of
agonists, representing a maximal or near-maximal activation of PLD and
other pathways. However, it does not necessarily represent the
physiological change needed for PA to exert its effect. Another
possible explanation is that cPA photolysis forms microdomains of high
PA concentration which are sufficient for causing its biological
effects.
)))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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