©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Release of Gelatinase A (Matrix Metalloproteinase 2) Induced by Photolysis of Caged Phosphatidic Acid in HT 1080 Metastatic Fibrosarcoma Cells (*)

(Received for publication, October 16, 1995)

Ben-Tsion Williger (1) Reuven Reich (2) Michal Neeman (1)(§) Tuvia Bercovici (3) Mordechai Liscovitch (1)(¶)

From the  (1)Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel, the (2)Department of Pharmacology, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, and the (3)Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Phospholipase D (PLD) (^1)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.


EXPERIMENTAL PROCEDURES

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(3), and 2-nitroacetophenone were from Aldrich. MnO(2) (activated grade), MgSO(4), and Silica Gel 60 aluminum thin layer chromatography (TLC) plates were purchased from Merck. [P]ATP and [^14C]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 H(2)O. Anhydrous MgSO(4) (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(2). 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(f) about 0.56) was scraped, and the cPA was extracted with methanol and quantitated by a phosphate assay as described before(13) .

Preparation of P- and ^14C-Labeled cPA

PA labeled with P was prepared from diacylglycerol to which P was transferred from [-P]ATP by diacylglycerol kinase as described previously(14) . Mixing the P-labeled or ^14C-labeled PA with PA dissolved in chloroform was followed by desalting as described above. [P]cPA and [^14C]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(3) 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/NH(4)OH (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.


RESULTS AND DISCUSSION

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(2) 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(2) 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. [^14C]cPA was loaded on a TLC plate and then ultraviolet illuminated for 1 min (right lane). A nonphotolyzed sample of [^14C]cPA was added (left lane), and the plate was developed with CHCl(3), 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(3)-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(3) 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(3) and CH(2) 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 times 10^5 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. (^3)


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 µMP-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(4)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.^3 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.^3



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 times 10^5 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 times 10^5 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.


FOOTNOTES

*
This research was supported in part by grants from the Israel Ministry of Science and Arts and the Deutsches Krebsforschungszentrum (Heidelberg) (to M. L.) and from the Clive and Judy Callman Fund for Cancer Research (United Kingdom) (to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Incumbent of the Dr. Phil Gold career development chair in cancer research.

Incumbent of the Shloimo and Michla Tomarin career development chair in membrane physiology. To whom correspondence should be addressed. Tel.: 972-8-342-773; Fax: 972-8-344-116; lhliscov@weizmann.weizmann.ac.il.

(^1)
The abbreviations used are: PLD, phospholipase D; BSA, bovine serum albumin; PA, phosphatidic acid; cPA, caged phosphatidic acid; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; MMP-2, matrix metalloproteinase 2.

(^2)
B.-T. Williger and M. Neeman, unpublished results.

(^3)
B.-T. Williger, unpublished observations.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.