Novel Proliferative Effect of Phospholipase AS in Swiss 3T3 Cells via Specific Binding Site*

Phospholipase A2 (PLA2), EC 3.1.1.4, which catalyzes the release of free fatty acids from the sn-2 position of glycerophospholipids, has been extensively studied from the viewpoint of eicosanoid production (Arita, H., Nakano, T., and Hanasaki, K. (1989) Prog. Lipid Res. 28, 273-301). Several lines of evidence suggest that extracellular PLA2 is pathophysiologically related to some disorders, including inflammation and hypersensitivity. Despite this, little is known of the precise mechanism of the pathological processes as well as their intrinsic correlation with dysfunction. Here, we report a novel PLA2 action on the proliferation of Swiss 3T3 fibroblasts via specific binding sites of approximately Mr 200,000. Pancreatic type PLA2 in the active form specifically recognized the sites and stimulated thymidine incorporation in DNA. Its inactive zymogen and other PLA2s from platelets, snake, and bee venoms showed much lesser activities. Although the physiological significance remains to be identified, our finding is the first to offer a new viewpoint on the effect of mammalian extracellular PLA2 on cellular function.

. Several lines of evidence suggest that extracellular PLAz is pathophysiologically related to some disorders, including inflammation and hypersensitivity. Despite this, little is known of the precise mechanism of the pathological processes as well as their intrinsic correlation with dysfunction. Here, we report a novel PLAz action on the proliferation of Swiss 3T3 fibroblasts via specific binding sites of approximately M , 200,000. Pancreatic type PLAz in the active form specifically recognized the sites and stimulated thymidine incorporation in DNA. Its inactive zymogen and other PLAzs from platelets, snake, and bee venoms showed much lesser activities. Although the physiological significance remains to be identified, our finding is the first to offer a new viewpoint on the effect of mammalian extracellular PLAz on cellular function.
In several inflammatory regions, levels of extracellular PLA2' activity are described to be elevated, which has been thought to play an important role in mediating some inflammatory processes (1, 2). Mammalian extracellular 14-kDa PLA2s described thus far can be classified into two types, group I (PLA,-I) and group I1 (PLA2-II), based on their primary structures (3). Several studies have implicated the correlation of PLA2-I1 in the pathogenesis of inflammation (4,5). We have recently found that some inflammatory factors dramatically increased PLA2-I1 secretion from several tissues of rat via enhancement of gene transcription (5)(6)(7)(8)(9). On the other hand, PLA2-I is mainly secreted from the pancreas as an inactive zymogen, which is further converted into the active form by protease and then has been thought to act as a digestive enzyme (10). However, recent studies have shown that PLA2-I is not restricted to the pancreas only (11). It was detected in human lung (12), rat stomach, and spleen (13,14), as well as in the human serum (15), which suggests some * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. distinctive roles for this type of enzyme. With the use of Swiss 3T3 fibroblast cells and PLA2-I from several animal species, we have now obtained evidence for the new type of function of PLA2-I, possibly unrelated to its phospholipid-hydrolyzing properties. In this study we characterized the PLA2-I-specific binding site and examined its correlation with physiological properties.

EXPERIMENTAL PROCEDURES
Materials-Porcine PLAz-I was purified to homogeneity according to the method of Puijk et al. (16). Human mature PLAz-I and its proform were purified from pancreatic juice (17), and recombinant human PLAz-I and its pro-form produced by Saccharomyces cereuisiae (18) were also used. Rat mature and pro-form of PLAZ-I were isolated from pancreas homogenate according to the method of Ono et al. (19). Rat and rabbit group I1 PLAzs were respectively purified from platelets as described previously (20,21). Purified PLAZs showed a single band of approximately 14 kDa by silver staining on SDSpolyacrylamide gel electrophoresis and high specific activities (50,000-70,000 nmol/min/mg of protein) using [3H]oleic acid-labeled Escherichia coli as substrate (22). Human and rat pro-PLA2-I showed weak fatty acids liberating activities but turned into much higher specific activities after treatment with trypsin. Other PLAzs from bee venom, Naja nnja venom, and Crotalus adamanteus venom were purchased from Sigma. Swiss 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 pg/ml streptomycin in a humidified atmosphere of 5% CO, and 95% air at 37 "C.
Iodination of Phospholipase A,-Iodination of PLAz-I was carried out by using a chloramine-T method (23). '2sI-Labeled sodium (0.5 mCi, Amersham Corp.) was added to porcine PLA2-I (20 pg) in 150 pl of 20 mM NaH2P0,, pH 7.5, and the reaction was initiated by adding 50 pl of chloramine T (9.6 mg/ml). After 20 s of incubation at room temperature, the reaction was terminated by adding 200 pl of Na2SZOs solution (4.8 mg/ml). The reaction mixture was then loaded on a Sephadex G-25 column (100 X 5 mm) for separation of lZ5I-PLAZ-I. The specific activity obtained was about 450 cpm/fmol. The iodinated ligand retained the same enzymatic activity with the native Binding Experiments-Confluent 3T3 cells grown in 35-mm diameter dishes were washed twice with phosphate-buffered saline and then incubated with various concentrations of lZ5I-PLA2-I in 1 ml of the binding medium (Hanks' medium, pH 7.6, containing 0.1% bovine serum albumin) for 2 h at 4 "C. After incubation, cells were extensively washed with phosphate-buffered saline, and cell-bound radioactivity was determined. The specific binding is defined by subtracting the nonspecific binding, the amount of '251-PLAz-I bound in the simultaneous addition of the unlabeled porcine pancreatic PLA, (500 nM), from the total binding. Competition experiments on 3T3 cells were performed by incubating with 1 nM 'zsII-PLA2-I in the presence of various concentrations of PLA2s at similar binding conditions. The IC, value was evaluated from the inhibition curves as the concentration which inhibits half of the 'zsII-PLA2-I specific binding.
Affinity Cross-linking Experiment-Confluent 3T3 cells grown in 150-mm diameter dishes (1.2 X lo7 cells) were incubated with 2 nM Cell Proliferation by Phospholipase AP

RESULTS AND DISCUSSION
When 12sII-PLAz-I (porcine) was incubated a t 4 "C with Swiss 3T3 fibroblasts, it could bind specifically in a saturable manner. The specific binding of 12"I-PLAz-I reached equilibrium after 2 h and was stable for up to 2 h at 4 "C. Although about a two times higher level of binding was detected at 37 "C than at 4 "C, further binding experiments were performed at 4 "C to avoid internalization of the binding site as well as binding to the newly synthesized site. 10" cells (Fig. 1B). The relative inhibitory effects of various phospholipases on lZsI-PLAz-I equilibrium binding were examined, and the concentrations which inhibit half of the PLAz-I specific binding (ICso) were summarized in Table I. The ICso value of unlabeled porcine PLAz-I corresponded well to the K d value. Rat and human PLAz-I showed almost the same ICso values with porcine PLAz-I, whereas the proenzymes of human and rat PLAz-I, PLA2s-I1 purified from rat and rabbit platelets, and toxic PLAzs from snake or bee venoms could not suppress the ligand binding at concentrations greater than 100 nM, demonstrating the specificity of the PLA2-I binding site for the mature type of PLA2-I derived from mammalian pancreas.
Identification of the binding proteins responsible for "' 1-PLAz-I binding was achieved by cross-linking experiments using a bifunctional cross-linker, DSS. l2'1-PLA2-I was bound  to Swiss 3T3 cells a t 4 "C, treated with 0.15 mM DSS, and then analyzed by polyacrylamide gel electrophoresis in the presence of SDS. As shown in Fig. 2, a single band at an apparent M , of 210,000 was detected only in the treatment with DSS, while formation of the cross-linked complex was completely blocked by the presence of excess unlabeled PLA2-I during the 1251-PLAz-I binding. Under nonreducing conditions, the same M , position was specifically labeled (data not shown). Assuming that the 12sI-labeled complex contains a singre molecule of both receptor and PLAz, subtracting the mass of the PLAz-I (14 kDa) suggests that the binding site for PLAz-I has a mass of approximately 200 kDa.
Lambeau et al. (25) have recently reported the specific binding protein in rat synaptic membranes which was recognized by neurotoxic snake venom PLAzs, while mammalian PLAz does not recognize this binding site. Thus, the PLAZ-I binding site we characterized in this study might differ significantly from the neurotoxic PLAz binding site.
As one of the physiological functions of the PLAz-I binding site in Swiss 3T3 cells, we found the effect of PLAz-I on ['HI thymidine incorporation into acid-insoluble DNA. When quiescent cells were incubated with PLAz-I alone for 24 h, DNA synthesis of 3T3 cells was stimulated in a dose-dependent manner as shown in Fig. 3. This effect of PLAz-I was synergistically enhanced in the presence of 100 nM insulin, which by itself showed weak mitogenic activity. Similar synergistic effects of PLAz-I were observed in the presence of platelet-derived growth factor (2 ng/ml) or endothelin (10 nM). The mitogenic effects of PLA,-I could not be affected by the treatment with indomethacin, demonstrating a direct effect of PLA2-I without any involvement of the growthpromoting prostanoids in Swiss 3T3 cells (26). Similar proliferative effects were observed in the treatment with rat and arachidonic acid. Neither liberation of free fatty acids nor release of lactate dehydrogenase was detected up to 1 PM concentration of both enzymes treated for 24 h, although 5 PM A23187 liberated a large amount of labeled fatty acids (data not shown). These data demonstrate that binding as well as proliferative effects of PLA2-I can be clearly distinguished from its phospholipid hydrolyzing activity, and the absence of*binding affinity of PLAZ-I1 cannot be attributed to its direct effect on the membrane phospholipids. Other evidence from the binding profile of PLA2-I, showing it not to be affected in the presence of EDTA (10 mM), further supports this specificity, because phospholipid hydrolysis by PLA2-I completely depends on submillimolar amounts of calcium (28). Recently, we found the same PLA2-I binding sites in some tissues of several animal species, especially in vas-culature. These findings suggest some role of PLA2-I in vascular function, which is now under further investigation. In conclusion, our finding provides a new aspect of phospholipase A2 in the modulation of cellular functions.