CD39 as a Caveolar-Associated Ectonucleotidase

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Biochemical and Biophysical Research Communications 262, 596 –599 (1999) Article ID bbrc.1999.1254, available online at http://www.idealibrary.com on

CD39 as a Caveolar-Associated Ectonucleotidase A. Kittel,* ,1 E. Kaczmarek,† J. Sevigny,† K. Lengyel,* E. Csizmadia,† and S. C. Robson† *Department of Neuroendocrine Cell Biology, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, 1450 Budapest, Hungary; and †Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Received July 26, 1999

CD39 is a human lymphoid cell activation antigen, (also referred to E-ATPDase or apyrase) that hydrolyzes extracellular ATP and ADP. Although it has been widely studied, its physiological role, however, still remains unclear. This ectonucleotidase generally is said to be evenly distributed in the membrane of the cells. However, we observed that in cell types which possess caveolae, specialised membrane invaginations involved in signalling, CD39 is preferentially targeted to these membrane microdomains. Since all molecules involved in signalling (eNOS, G-proteins, receptors) which are targeted to the caveolae undergo posttranslational modifications (e.g., palmitoylation) we hypothesize the same to be the case for CD39. Furthermore, its presence in the caveolae supports its participation in signalling events. © 1999 Academic Press

Since 1957, the first time when the term “ectoATPases” was used (1), more than forty years have passed, however, their physiological role remains unclear. CD39/ATPDase, an integral membrane enzyme, and one of the most intensively studied members of this enzyme family, is considered to play an important role in purinergic signalling, in thromboregulation and in neuroprotective processes by converting ATP released by damaged cells. We have recently observed that CD39 appears to be preferentially targetted to the caveolae of endothelial cells. Caveolae are specialized detergent-insoluble plasmalemmal micro-domains that are known for about 30 years. They have been the focus of intensive research during the past several years. Since caveolae 1 To whom correspondence should be addressed. Fax: 361 3134 630. E-mail: [email protected] Abbreviations used: ecto-NTPDase, ecto-nucleoside-triphosphatediphosphohydrolase; E-ATPDase, ecto-adenosine-triphosphate-diphosphohydrolase; E-ATPase, ecto-adenosine-triphosphatase; HUVEC, human umbilical vein endothelial cell; eNOS, endothelial nitrogenmonoxide synthase.

0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

are suggested to be involved not only in the transcytosis of macromolecules, but especially in the regulation of signal transduction (2, 3), there arises the possibility that ectonucleotidases, targeted to these special membrane parts, are also participants in signalling events. This role had been suggested in several works on ecto-ATPases (e.g., 4 –10), but the role of caveolar targeting of CD39 has not been demonstrated. To study the CD39/ATPDase localization in caveolae, we used a human umbilical vein endothelial cell culture (HUVEC) which possesses native ectonucleotidase activity and a COS-7 epithelial renal fibroblast cell culture which has this activity after transfection whith CD39cDNA. Both cell types showed caveolae-like invaginations in their plasmalemmal membrane. Caveolae were identified with anticaveolin immunostaining in HUVEC cells. Enzyme histochemical staining for e-ATPDase activity as well as immunostaining for CD39 showed the same localization and special role of the caveolae in the distribution of the enzyme. MATERIALS AND METHODS Reagents. All reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified. Preparation of cell cultures. Preparation of HUVEC cell cultures and transient transfection of COS-7 cells with CD39cDNA were performed as described by Kaczmarek and colleagues (11). The transfected cells were fixed for enzyme and immuno histochemistry on the third day after transfection. HUVEC from fresh umbilical veins were cultured in M199 with 20% FCS, heparin (100 mg/ml), and endothelial growth factor (50 mg/ml, BioWhittaker, Walkersville, MD); COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS. Both media were supplemented with L-glutamine (2 mM), penicillin G (100 units/ml) and streptomycin (100 mg/ml) (11). All cells were grown in culture dishes at 37°C in a humified incubator with a 5% CO 2 atmosphere. Cultured cells were harvested by scraping. Cells for electron microscopic investigations were transferred to 8 well plastic chamberslides (Nunc, Naperville, IL) two days before fixation. Enzyme histochemistry. For demonstration of e-NTPDase (ectoATPDase) activity, a cerium precipitation method was used as de-

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scribed by Kittel (12). After discarding the medium and washing with cacodylate buffer (0.25 M sucrose in 0.05 M Na-cacodylate, pH 7.4) the cells were fixed with icecold cacodylate buffer containing 3% paraformaldehyde (Merck, Darmstadt, Germany), 0.5% glutaraldehyde (Taab, Aldermaston, Berks, UK) and 2 mM CaCl 2, (pH 7.4), for 30 min. After several rinses in 0.07 M Tris-maleate buffer, pH 7.4, the cells were incubated in a medium containing, ATP or ADP (1 mM) as substrate, 2 mM CeCl 3 (capturing agent for the liberated phosphate), 1 mM levamisole (inhibitor of alkaline phosphatases, Amersham, Buckinghamshire, UK), 1 mM ouabain, (Na 1/K 1-ATPase inhibitor, Merck, Darmstadt, Germany), 50 mM a,b-methylene-ADP (59-nucleotidase inhibitor) and KCl (5 mM), in Tris-maleate buffer (70 mM, pH 7.4) for 30 min at 37°C. Incubation was followed by three rinses in Tris-maleate buffer. The samples were postfixed for 30 min in 1% OsO 4 (Taab, Aldermaston, Berkshire, UK) dissolved in cacodylate buffer, dehydrated and embedded in Epon (Fluka, Buchs, Switzerland). Ultrathin sections were cut and examined in a Hitachi 2001 transmission electron microscope (Hitachi Corp., Japan). To demonstrate the specificity of the reaction product, in control experiments substrate (ATP or ADP) was omitted from the incubation medium. Immunostaining with a-CD39 (Accurate, CA) and a-caveolin (Santa Cruz Biotechnology, CA) monoclonal antibodies. Fixation was carried out as above. After rinsing with PBS, the cells were blocked with 5% NHS in PBS for 30 min and incubated overnight with the antibodies (CD39 antibody in 1:500 dilution, a-caveolin in 1:50 dilution) on a shaking plate at 4°C. ABC method was used and VectorVIP as chromogen (Vector Laboratories, Inc., Burlingame, CA) was applied according to the manufacturer’s instruction. Postfixation, embedding and electron microscopic investigations were done as in the case of enzyme histochemical staining.

RESULTS Ecto-NTPDase (ATPDase) activity of both HUVEC and CD39cDNA transfected COS-7 cells was present as cerium phosphate deposit at exactly the same place as the phospate liberated from the extracellular ATP or ADP during hydrolysis. While native COS-7 cells did not show almost any ecto-enzyme activity (Fig. 1a), the CD39 cDNA transfected cells had strong ecto-ATPase activity which seemed to be evenly distributed over the cell membrane. Some caveolae-like structures also were filled with the cerium phosphate deposit, indicating the presence of the enzyme (Fig. 1b). When ADP was used as substrate, the distribution of the cerium phosphate precipitate was similar, including the caveolae (Fig. 1c). Immunostaining for CD39 showed the same localization both in the cell membrane and in caveolae as the enzyme activity and proved the identity of the ectoATPDase enzyme with CD39 (Fig. 1d). HUVEC cells possess native ecto-NTPDase activity. Control experiments for enzyme histochemical staining, made by incubating the cells in the absence of ATP (Fig. 2a), were negative. When the incubation medium contained ATP as a substrate, ecto-ATPDase activity was present in the cell membrane and in many caveolae (Fig. 2b). Staining with a-CD39 gave the same result (Fig. 2c). Immunostaining for caveolin proved that the mem-

FIG. 1. (a) Native ecto-ATPase activity in COS-7 cell. Almost no cerium deposit in the ramified caveolae indicated by empty arrow. (b) Ecto-ATPase activity in CD39cDNA-transfected COS-7 cells. Caveolae are full with cerium phosphate precipitate. Cell membrane also shows ecto-ATPase activity. (c) Ecto-ADPase activity in CD39cDNAtransfected COS-7 cells. (d) Immunostaining for CD39 in CD39cDNAtransfected COS-7 cells. The staining is exactly the same as in the acse of ecto-ATPDase activity.

brane invaginations and the small vacuolae in HUVEC cells, are caveolae (Fig. 2d). They are morphologically identical to those found in COS cells.

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pressed CD39/ecto-ATPDase as was demonstrated by both enzyme and immunohistochemical methods. The caveolae of the cell cultures showed a high concentration of the ecto-enzyme. This finding is in agreement with our previous observation where the caveolae of different cell types also showed high ecto-ATPase activity. Interestingly, this caveolar activity was significantly stronger in activated cells: in elicited macrophages (13), in concanavalin A-treated smooth muscle cells (14) or in lipopolysaccharide-treated capillary endothelial cells (12). Another observation was that not only the ectoATPase activity but also the number of caveolae depended on the condition of the cell. Activated cells contained much more caveolar structures, proving their significance as dynamic pieces of the plasma membrane. Also it seemed to support their hypothesized role as coordinators of the interactions of the cell with its microenvironment (3). The main structure of caveolar membrane has already been clarifiedIt markedly improved our understanding of how caveolae can be a “message center” of the cells possessing them (15). Recent studies revealed that caveolae serve as docking places in the plasmalemma for numerous proteins involved in signal transduction (e.g., G-proteins, eNOS, tyrosine kinases, phospholipase D1 (2, 16–27). All protein molecules involved in signalling, have to undergo posttranslational modification to target them to the caveolae and unable to them to interract with the scaffolding domain of caveolin, the major structural component of the caveolae membrane (17, 19–21, 23, 27, 28). Since we hypothesize that the targeting to the caveolae is also the result of posttranslational modifications, the membrane bound and caveolar localized ecto-ATPDase should be different in both structure and function. The presence of CD39/ATPDase in these membrane invaginations and the elucidation of the targeting pathway, may support the special role of caveolae in endothelial cells as well as clarify the biological role of CD39. The high level of this protein in the “message center” of several cell types, and its higher concentrations after some stimuli or inflammation, may be of great importance to help the cell to optimize its responses according to the actual spatio-temporal signalling. ACKNOWLEDGMENTS FIG. 2. (a) HUVEC cells, control staining for ecto-ATPase activity. No precipitate in the caveolae (empty arrow). (b) Caveolae and the cell membrane in the HUVEC culture demonstrate ecto-ATPase activity. (c) Immunostaining for CD39 in the caveolae in HUVEC. (d) HUVEC-caveolin staining. Anti-caveolin labels the same invaginations and vesicle-like structures as CD39 antibody. Bars indicate the same distance in all figures: 250 nm.

This study was aided by Hungarian OTKA Grant T019860. The authors thank Professor Henry Teuchy (LUC, Belgium) for the critical reading of the manuscript and Mr. Gyozo Goda for the excellent technical assistance.

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DISCUSSION Endothelial (HUVEC) and epithelial (CD39cDNA transfected renal fibroblast COS-7) cell cultures ex598

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