1082(1991) 285-292 ~. 1991 ElsevierSciencePublishersB.V.0005-2760/91/S03.50 ADONIS 0005276091001~.18

285

Biochimica et Biophysica A cta.

BBALIP53624

Intracellular localization of group II phospholipase A 2 in rat vascular smooth muscle cells and its possible relationship to eicosanoid formation Hidetake Kurihara, Tohru Nakano, Nobuo Takasu and Hitoshi Arita Shionogi Research Lahoratortex. Shionogt & Co.. Ltd. FuktL~hima-ku. Osaka (Japan)

(Received 23 July ITS90) (Revised manuscriptrecetved 20 November 19q0)

Key words: PhospholipaseA,; Eicosanoidformation; Immunofluore.~encc:lmmun~elcctronmicr(~,copy:(Rat vascularsmooth muscle cell) We investigated the localization of group !! phospholipase A 2 (PLA z-ll) in rat vascular smooth muscle cells (VSMCs) by applying immunofiuorescence and immunoelectron microscopy with its polyclonal antibody. In unstimulated cells, no immonolabelling was detected in the cells. On the other hand, in the cells stimulated v~ith tumor necrosis factor (TNF) a n d / o r forskolin (FK), intense fluorescence was detected in the cytophtsm. The inununoperoxidase reactions were detected in the cisternae of rough endoplasmic reticulum (rER), trans-cisternae of Golgi apparatus, and small vesicles beneath the plasma membrane. Western blot analysis showed VSMCs secrete PLA2-11 alter .stimulation. Secreted PLA,-I! was associated with the plasma membrane and extracellular matrix. Colchicine inhibited PLA z-II syathesis and its secretion to the extracellular space. These observations indicate that in VSMCs PLA 2-11 is synthesized at rER, transported to Golgi apparatus, discharged into extracellular space via the small vesicles, and microtulmles may concern with its process. Furthermore, in VSMCs treated with TNF or TNF + FK, prostaglandin Ez formation was also increased. Acfinomycin D and cycloheximide inhibited the potentiation oi the prostaglandin E2 formation induced by TNF or TNF + FK, indicating that both RNA and protein synthesis are required for the potentiation. These results suggest an involvement of PLA 2-11 in the prostaglandin formation.

Introduction Phospholipase A 2 (PLA 2) specifically releases fatty acids esterified at the sn-2 position of glycerophospholipids. Since arachidonic acid, a precursor of prostaglandins and leukotrienes, is normally found at the sn-2 position, PLA 2 action is thought to be the ratelimiting step in the production of the eicosanoids [1]. Vadas and Pruzanski [2] have recently reviewed the involvement of secretory PLA2 in pathological

PLA.,-II. group ll phospholipase A2; VSMC, vascular smooth muscle cell; TNF, tumor necrosis factor; FK. forskolin; rER. rough endoplasmic reticulum; DMEM. Dulbecco's modification of Eagle's medium; BSA. bovine serum albumin; PBS. phosphate-buffered saline; ECM, extracellular matrix. Abbreviations: PEA 2. phospholipase A2;

Correspondence: H. Arita. Shionogi Research Laboratories, Shionogi&Co., Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553. Japan.

processes, especially those of inflammation. Mammalian PLA~ characterized thus far can be classified into two groups based on the primary structure. Group I PLA, exists mainly in the pancreas [3-51. However, group 11 PLA, (PLA2-11) is found in the extracellular spaces of some inflammatory regions [6-8], and therefore is thought to participate in the pathogenesis of inflammatory diseases. Our recent study using cultured rat vascular smooth muscle cells (VSMCs) demonstrated that two inflammation-related cytokines, tumor necrosis factor (TN F) and interleukin 1. and cAMP.elevating agents such as forskolin (FK) induced PLA2-11 secretion from the cells by stimulating the synthesis of PLA2-11 mRNA [9]. We also feund that glucocorticoid, a potent antiinflammatory agent, inhibiled the secretion of PLA2-11 [10]. These findings further support the idea that the secretion of PLA2-Ii may be important for the progress of inflammation. The de novo synthesized PLA2-11 seems to be rapidly secreted by the cells, although the secretory process remains obscure. Not only the secretory

286 process for the PLA2-1I, but also its subcellular localization elicits our interest fr6m the viewpoint of eicosa,:oid formation within the cells. In th- present study, we investigated the secretory process of PLA 2-II and its intracellular localization in rat VSMCs using immunocytochemical techniques. We also examined the relationship bctwccn PLA2-II and eicosanoid formation in stimulated cells.

recognized only protein band with approx. Mr 14 kDa, which was identical with authentic rat PLA~-II.

Experimental procedures

Immunofluorescence

Materials Human recombinant TNF was purchased from Genzyme. Other chemicals were from Sigma.

Cell:; Rat VSMCs were isolated by enzymic digestion of media of thoracic aorta from male Sprague-Dawley rats as described by Chamley-Campbell et al. [111. The cells werc cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal calf serum. Confluent VSMCs in 35-mm-diameter dishes were washed twice with DMEM and incubated Ioi 24 h with 1.5 ml of DMEM coniaining 0.1 m g / m l bovine serum albumin (BSA) with or without the stimuli. At the end of the incubation, the cells were washed and used for immunocytochemistry. 1/50th each of the medium and the cells in a 35-mm-diameter dish were subjected to Western blotting.

PLA 2 activi O' assay Phospholipase activity was measured by the hydrolyof {-~H]oleic acid-labeled (Amersham Corp.) Escherichia colt phospholipids as described elsewhere

sis

[10].

Western blotting Samples were separated by SDS polyacrylamide gel electrophoresis, and immune,blotting with the anti-rat PLA2-11 IgG was done t, si,lg a Blotting detection kit (Amersham).

VSMCs were cultivated with TNF (200 units/ml) a n d / o r FK (10 #M), or without the stimuli on tissue culture chamber slides (Lab-Tek) for 24 h. Cells were rinsed in phosphate-buffered saline (PBS) and fixed with chilled acetone for 5 rain. Air-dried cells were then rinsed in PBS five times for 5 min each and incubated with anti-PLA,,.-I1 IgG (diluted in PBS containing 1% BSA) for 2 h at room temperature. They were rinsed in PBS three times and incubated with fluorescein isothiocyanate-labeled goat anti-rabbit IgG (ICN) in PBS for 1 h at room temperature. For visualizing of actin filaments, fixed cells were stained with rhodaminelabeled phalioidin (Wako, diluted 1 : 100 in PBS) for 45 min at room temperature. After washing with PBS three times, stained preparations were mounted with glycerinPBS (1:1) and examined using an FX-S RFL fluorescence microscope (Nikon).

lmmunoperoxide staining Cells were fixed in a periodate-lysin-paraformaldehyde fixative at 4°C for 3 h and then washed in PBS five times for 10 rnin each. Cells were then incubated for 15 min at room temperature with the permeabiliza-

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Prostaglandin E, formation At the end of the 24 h incubation, the cells in 35-ram-diameter dishes were washed two times with DMEM containing 0.1 m g / m l BSA and incubated for I0 rain with or without 1 unit/ml thrombin in 1.5 ml of DMEM containing 0.1 m g / m l BSA. P ostaglandin E 2 released into the medium was measured with a prostaglandin E 2 radioimmunoassay kit (New England Nuclear).

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Antibody Polyclonal rabbit anti-rat PLA 2-11 lgG was produced against PLA,-I! released from thrombin-stimulated rat platelets, which was purified as previously described [12]. The antibody specifically recognized PLA2-11 and did not cross-react with rat pancreatic group i phospholipase A, as described elsewhere [13]. Immunoblotting analysis data in the culture medium and the cells of V:SMCs (see Fig. 1) also indicate that the antibody

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Stimulation-induced increas~ of PLA;-I! in culture medium

and in VSMCs. PLA:-II in the culture medium(M) and in the cells (C) of VSMCsincubatedwithout stimuli for 24 h (Control)or VSMCs incubated with TNF (200 units/ml) plus FK (10 #M) for 24 h (TNF+ FK) was analyzed by immunoblottingusing anti PLA2-11 antibody as described under F.xpcrimentalprocedures. The molecular weight markers ovalbumin (46000), carbonic anhydrase (30000). trypsin inhibitor(21500), lysozyme(14 300) and aprotinin(6 500) were run as standards as indicatedby arrow.

287 tion buffer containing 0.01% saponin and 0.1% BSA in PBS. The samples were incubated ha anti PLA2-II lgG in PBS containing 0.1% BSA for 2 h at room temperature. After rinsing in PBS for 30 min, cells were stained by the avidin-biotin-peroxidase complex method using Vectastain ABC KIT (Vector Laboratories). The materials were incubated with biotinylated anti-rabbit lgG for 1 h at room temperature. After washing three times with PBS, the ceils were made to react with avidin-biotinperoxidase complex for 30 min at reom temperature. The stained samples were washed for 30 rain with PBS and fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), for 1 h at room temperature, and further washed five times with 0.1 M sodium cacodylat,: buffer (pH 7.4) (10 min each). After rinsing in 50 mM Tris-HC! buffer (pH 7.6), the samples were incubated with 0.2% diaminobenzidine hydrochloride,

0.01~ !-1_~O_,and 50 mM Tris-HCl (pH 7.6), foi viewing. After washing in PBS three, times (10 rain each), the stained cells were postfixed in 1% O,O4 in 0.1 M sodium cacodylate buffer (pH 7.4). The fixed specimens were washed with 0.1 M sodium cacodylate buffer (pH 7.4), containing 7~i; sucrose, for 30 min, dehydrated with a graded concentration of ethanol and embedded in Epon epoxy resin. Ultrathin sections were stained with lead citrate and examined with a JEOL 100CX electron microscope. Results

Western blotting analysis I n the previous paper I91, we demonstrated that TNF and FK stimulate the synthesis of PLA,-II via two different mechanisms in rat VSMCs and that they act

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Fig. 2. lmmunofluorescence micrograph of PLA2-ll in rat VSMCs. Cells were incubated with T N F (200 units/ml) ~a), FK (10 pM) (b), or TNF plus FK (c) for 24 h at 37°C. After stimulation with TNF or FK, dot-like labeling was observed in the cytoplasm, especially in the perinuclear space. In the cells stimulated with TNF plus FK. strong labeling was seen in the whole cytoplasm. No fluorescence localization was evident in the control cells (d). x 250.

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Fig. 3. Alterationsof actin filamentsbundle organizationin rat vascular smoothmusclecells. Cellswere fixedand stained with rhodamine-phalloidin for visualizationof F-actin. Actin filamentbundles were clearly observedin control (d) and TNF-treated cells (a). On the other hand, actin bundleswereremarkablydecreasedin FK-treated(b) and TNF + FK-treated cells(c). x 250.

synergistically. The stimulated VSMCs secreted PLA2-1I into extracellular medium. In order to clarify what part of the PLA2-1I synthesized in the cells was secreted, we used immunoblotting to compare the amount of PLA 2-II associated with the cells and that in the medium (Fig. 1). Although a large part of the PLA2-1I which was synthesized upon stimulation with T N F + FK was secreted from the cells, an increase of intracellular PLA2-11 was also detected.

hblnllolofhtores('ePlc'e VSMCs were incubated with TNF a n d / o r FK or without them for 24 h, and the distribution of PLA2-11 was examined by immunofluorescence (Fig. 2). Control cells, showing a spindle-shape, were not detected in the positive reaction against anti PLA2-11 lgG. The cells

treated with T N F showed features similar to the control cells. FK treatment caused morphological change leading to rounding of cells. FK treated cells contained nu,,~erous long extensions. The cells stimulated with TNF or FK were labeled with anti PLA2-11 IgG. Positive materials showed dot-like patterns in the cytoplasm, especially in the perinuclear space. On the other hand, most of the cells treated with T N F + FK changed morphologically to display features similar to those treated with FK, and the entire cytoplasm containing the long extensions of these cells were strongly positive to the anti-PLA2-11 IgG. VSMCs were studied with rhodamine-phalloidin for visualization of polymerized actin filaments (Fig. 3). Actin-containing stress fibers were abundant in control and TNF-stimulated VSMCs. Actin filament bundles

289 were distributed along the long axis. In contrast, actin bundles were remarkably decreased in the cytoplasm of FK- treated cells, and only microfilaments associated with the plasma membrane were seen. The cells stimulated with both FK and TNF, which changed morphologically, showed the similar pattern to that of the cells stimulated with FK on the rhodamine-phalloidin staining.

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We also investigated the localization of PLA: at the electron microscopic level using the immunoperoxidase technique. In the cells stimulated with both TNF and FK, the plasma membranes facing the plastic dish were prominently stained with anti-PLA,-ll IgG (Fig. 4). On the other hand, the apical membranes facing the medium were mostly negative to immunoreaction. PLA ,-II was also found in small vesicles, which were scattered beneath the basal plasmalemma (Fig. 5a). The membranes of small vesicles were densely labeled, but the central parts of the cavities were free from immunolabeling. The small cavities of basal plasma membrane were positive, but the reaction product was very scarcely detected in those on the apical membrane (Fig. 4, 5b). The rough endoplasmic reticulum (rER) and the Golgi apparatus of the cells used in this experiment were well developed, which means that the cells were in the synthetic phase. In some of the cells stimulated with TNF + FK, the whole cavity of the rough endoplasmic reticulum cisternae was heavily labeled (Fig. 6e,). On the other hand, Golgi complex was partially stained. Especially the trans..cisternae of the Golgi complex were

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exclusively labeled (Fig. 6b). The peroxidase reaction was often observed in the multivesicular bodies. In the sections of unstimulated cells, no reaction products were observed on the plasma membrane or in the cell organelle containing rER, Golgi apparatus and small vesicles (data not shown). Effect of coichicine on the secretion of PLA ,-!I

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In order to investigate the involvement of cytoskeletal elements in the secretory processes of PLA,.-il, the effect of colchicine, an inhibitor of microtubule assembly, on PLAz-II secretion was studied. As shown in Table 1, colchicine treatment of VSMCs inhibited the secretion of PLA., induced by TNF or FK, suggesting the involvement of microtubules in the secretion pathway of PLA2-II. On the other hand, cytochalasin B which inhibits the assembly of microfilaments did not affect the secretion. If colchicine inhibited only the transport process of PLAz-II, the PLA,-II synthesized would accumulate in the cells. However, as no accumu-

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lation of PLA,-II in the cytoplasm was observed in the presence of colchicine, it seems that colchicine also inhibit the synthesis of PLA2-11 (Fig. 7).

Prostaglandin E. formation in VSMCs The results thus far indicated that the amount of

and FK, which raises the possibility that the stimulation potentiates eicosanoid formation in the cells. In order to check this, we examined prostaglandin E2 formation by stimulated and non-stimulated cells. VSMCs treated with TNF or TNF + FK for 24 h induced approx.

PLA~-II in VSMCs increased by stimulation with TNF

TABLE I

Effect of C,'to.~'k(,leton mhibitors on the release '4 P ¢.A.,-I! Rat VSMCs were stimulated with l0 pM FK or 200 units/ml T N F in the presence or absence of 10 #M cytochalasin B or 50 ttM colchicine. PLA: activity released during 24-h stimulation is expressed with the values without inhibitors as 100~.

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Fig. 7. Inhibition ol" P L A : synthesis of rat VSMCs by colchicine. Subconfluent VSMCs in 35-ram-diameter dish were stimulated with T N F + FK for 24 h in the presence or absence of 50 #M colchicine. Ceils were stained with anti-PLA.~-ll antibody by applying indirect immunoperoxidase method as described under Experimental procedures. Synthesis and secretion of P L A , - l l were remarkably inhibited by pletreatment ol" colchicine.

291 TABLE II

Enhanced production of prostaglandin E: h.t" VSMCs treated wtth TNt." or TNF pht~ FK

Rat VSMCswere treated with 200 units/ml TNF or TNF plus 10 ~.M I:K in the pre~ence or ahca.,nceof 1 .aM dexamethasone. I ~M actinomycin D. or 10 ktg/ml cycloheximide.The cells were then washed and stimulaied with 1 unit/ml thrombin for 1O rain. Prostaglandin E, released into the medium during the stimulation was measured. Inhibitors None Dexamethasone ActinomycinD Cycloheximide

Prostaglandin E2 (pg/ml) control "INF-treated TNF + FK-treated 12 103 840 l1 18 49 20 17 22 12 24 39

5-15-fold prostaglandin E~ formation compared with non-treated cells (data not shown). Furthermore, 2,l-h treatment of VSMCs with TNF or TNF + FK potentiated thrombin-induced prostaglandin E~ formation (Table II). These data suggest that PLA ,-II synthesized by the stimulations may directly couple with agonist-induced prostaglandin E 2 production. Actinomycin D, an inhibitor of RNA polymerase, and cycloheximide, an inhibitor of protein synthesis, inhibited the TNF- or T N F + FK-induceu potentiation, indicating that both RNA and protein synthesis were required for the potentiation: Furthermore, dexamethasone, a synthetic glucocortieoid, also inhibited it. These inhibitors also block synthesis of PLAz-ll in VSMCs [10], supporting the involvement of PLA,-II in eicosanoids formation. Discussion

This study shows that PLA2-1I can be immunocytochemically detected in the cisternae of rER, Golgi apparatus and small vesicles in the cytoplasm of stimulated VSMCs, but not in those of the control ones. A sequence study of rat PLA2-II eDNA has indicated that nascent rat PLA2-II protein possesses a signal sequence always found in common secretory proteins [14], suggesting that rat PLA2-11 is secreted through a pathway which ordinarily transports the secretory proteins. The present results clearly point to the pathway in which PLA2-11 seems to be synthesized at the rER, transported to the Golgi apparatus and secretory vesicles, and secreted into the ex.:racellular space upon stimulation with TNF and FK. Perhaps of even greater interest is the finding that PLA2-11 ;.s not associated with the membrane of r~R while it is associated with lhe membranes of Golgi apparatus and the secreto.,'y vesicles as well as the plasma membranes facing t~,e plastic dish. VSMCs u:;,:ally function as the contractile elements in normal v..~ssel wall. However, phenotypic change from a contractile

to a synthetic form occurs under abnormal conditions, i.e., atherosclerosis, or during culture [15-17]. Synthetic VSMCs secrete large amounts of extraceUular matrix (ECM) components including elastin, collagen, proteoglycans and fibron~tin. Those ECM components are also s'¢nthesized and secreted throug,h the rER-Golgi system [18-22]. Furthermore, cultured VSMCs tend to secrete the ECM components to the basolaterai membrane. Rat PLA~-II strongly binds to heparin 112], suggesting that it has an affinity for negatively charged polysaccharides. Golgi apparatus is a muhipotential organelle, which is involved in glycosylation, sulfation and phosphorylation of polypeptides [23]. Synovial ceils and chondrocytes as well as vascular smooth muscle cells, which secrete PLA, to the extracellular space, produce large amounts of the ECM. Megakariocytes also produce the proteoglycans and store those in the alpha-granules [24,251. Our immunocytochemicai study shows that alpha-granules of rat platelets are positive against anti-PLA,-ll antibody (unpublished data). Although it remains unknown that PLA,-II is modified in the Golgi apparatus, it is possible that PLA2-11 is associated with the extracellular matrix concentrated and sulfated in the Golgi apparatus. Our results on the intracellular localization of PLA:-ll suggest that PLA,II is transported together with the ECM components, and binds to the polysaccharides of membrane proteins and the ECM components. Our data indicate that FK induced the morphological change in VSMCs, which was tightly coupled with the disappearance of actin bundles. It has been reported that the elevation of intracellular cAMP levels induce the similar morphological change in several cell types such as fibroblasts [26], thyroid cells [27], smooth muscle cells [281, rheumatoid synovial cells [29]. Therefore, it is likely that the morphological change with the rearrangement of actin filaments of VSMCs after stimulation of FK is due to the elevation of intraceilular cAMP. Furthermore, our data suggest that FK plays a dominant role on the change of morphology with the reorganization of actin filame~t,s when VSMCs are stimulated with both TNF and FK at the same time. Cytochalasin B, an inhibitor of actin polymerization, did not affect the secretion of PLA2-11 in the cells stimulated with FK or TNF. Therefore, secretion of PLA,-II may not be affected by the rearrangement of actin filaments. In contrast, treatment of the cells ~,ith colchicine, a compound which inhibits the microtubul,: assembly, caused suppression of the PLA2-II secretion. This result raised the possibility that microtubules migl:, be involved in the intracellular transport of PLA2-Ii. ECM secretion is also known to be inhibited by colchicine [30,31]. However, the inhibitory mechanism of PLA2-11 secretion remains obscure since colchicine also blocked PLA,-II protein synthesis at the dosage used. This study offers the first evidence for a possible

292

correlation between eicosanoid production and the intracellular PLA,-II level with results for a specific antibody. Although most of the synthesized PLA2-II is secreted from the cells, it is ascertained that some part of the c~.zyme still reread,as in ;.he cell organella. Recently. Ono et al. [32] purified the PLAz-II with the same molecular species from membrane fraction of rat spleen. "r;~ese data suggest that PLAz-II may exist as tightly membrane-bound form in some tissues, whereas many problems remain to be solved. We are now conducting further investigation to confirm the intracellular function of PLA2-1I. References I Van Den Bosch, H. (1980) Biochim. Biophys. Acla 604. 191-246. 2 Vadas. P. and Pruzanski, W. (1986) Lab. Invest. 55. 391-404. 3 Scilhamer. J.J.. Randall. T.L., Yamamlka. M. and Johnson. L,K. (1986) DNA 5. 519-527. 4 Ohura. O.. Tamaki, M.. Nakamora. E.. Tsuruta, Y., Fujii. Y.. Shin. M., Teraoka, H. and Okamoto. M. (1986) J. Biochem. (Tokyo) 99, 733- 739. 5 Sakata. T.. Nakamura. E.. Tsurula, Y.. Tamaki. M.. Teraoka. H., Tojo. H.. Ono, T. and Okameto. M. (1989) Biochim. Biophys. Acta 1007. 124-I26. 6 Hara. S.. Kudo. !.. Matsuta. K., Miyamoto, T. and Inoue. K. (i988) J. Biochem. (Tokyo) 104. 326-328. 7 Seilhamer, J.J,. Pruzanski, W.. Vadas, P.. Plant, S.. Miller. J.A.. Kloss. J. and Johnson, L.K. (1989) J. Biol. Chem. 264. 5335-5338. Kramer. R.M.. Hession, C.. Johansen. B.. Hayes. G., McGray, P.. Chow. E.P.. Tizard, R. and Pepinsky. R.B. 0989) J. Biol. Chem. 264. 5768-5775. 9 Nakano. T.. Ohara. O.. Teraoka. H. and Arita, H. (1990) FEBS Lett. 261. 171-174.

10 Nakano. 1".. Ohara. O.. Tcraoka. H. and Arita. H. (1990) J. Biol. Chem. 265. 12745-12748. 11 Chamley-Campbell, J.. Campbell. G.R. and Ross R. {1979) Physiol. Rev. 59. 1-61. 12 Horigome K.. Hayakawa, M., Inotle. K. and Nojima, S. {1987) J. Biochem. (Tokyo) 101. 625-631. 13 Nakano, T, and Arita. H. (1990) FEBS Lett. 273.23-26. 14 ishizaki. J,. Ohara. O.. Nakamura. E., Tamaki. M., Ono, T.. Kanda. A., Yoshida, N., Teraoka, H.. Tojo. H. and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 162, 1030-1036. 15 Ross. R. and Glomset. J.A. {1973) Science 180. 1332-1339. 16 Gimbrone. M.A.. Jr. and Contran. R.S. {1975) Lab, Invest. 33, 16-27. 17 Campbell. G.R. and Campbell. J.H. (1985) Exp. Mol. Pathol. 42. 139-162. 18 Ross. R. and Klebanoff, S.J. {1971) J. Cell Biol. 50, 159-171. 19 Ross. R. {1971)J. Cell Biol. 50, 172-186. 20 Wight, T.N. and Ross. R. (1975) J. Cell Biol. 67, 675-686. 21 Gerrity, R.G., Adams. E.P. and Cliff. W.J. 0975) Lab. Invest. 32, 601-609. 22 Chemnitz. J. and Christensen. B.C. (1983) Eur. J. Cell Biol. 30. 200-204. 23 Farquhar, M.G. and Palade G.E. (1981) J, Cell Biol. 91, 77s-103s. 24 MacPherson G.G. (1972) J. Cell Sci. 10. 705-717. 25 Schick. B.P.. Walsh, C.J. and Jenkins-West. T. (1988) J. Biol. Chem. 263, 1052-1062. 26 Willingham. M.C. and Pastan. I. (1975) J, Cell Biol. 67, 146--159. 27 Westermark, B. and Porter, K.R. (1982) J. Cell Biol. 94. 42-50. 28 Smith. J.B. (1984) J. Cell. Physiol. 121,375-382. 29 Baker, D.G., Dayer, J-M., Roelke, M., Sehumachcr. H.R.. and Krane. S.M. (1983) Arthritis Rheum. 26, 8-14. 30 Ehrlich. H.P.. Ross, R., and Bornstein, P, (1974) J. Cell Biol. 62. 390-405. 31 Chaldakov. G.N. and Vankov, V.N. (1986) Atherosclerosis 61, 175-192, 32 Ono, T., Tojo. H., Kuramitsu. S., Kag~miyama. H. and Okamoto, M. (1988) J. Bioi. Chem. 263, 5732-5738.