(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) ()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.
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 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.
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 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.
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-CH of the ethyl ester
(J
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 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.
Next we have measured the incorporation of cPA into HT 1080
cells by introducing [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. (
)
Figure 3:
Uptake (A) and photolysis (B) of cPA in HT 1080 cells. A, serum-deprived cells were incubated with 16 µM
[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.
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-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).
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. 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.
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
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.
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.