Neutral ceramidase secreted by endothelial cells is released in part associated with caveolin-1

ABB Archives of Biochemistry and Biophysics 417 (2003) 27–33 www.elsevier.com/locate/yabbi

Neutral ceramidase secreted by endothelial cells is released in part associated with caveolin-1 Elena Romiti,a Elisabetta Meacci,a Chiara Donati,a Lucia Formigli,b Sandra Zecchi-Orlandini,b Marta Farnararo,a Makoto Ito,c and Paola Brunia,* b

a Dipartimento di Scienze Biochimiche, Universit
a degli Studi di Firenze, Viale G.B. Morgagni 50, 50134 Firenze, Italy Dipartimento di Anatomia, Istologia e Medicina Legale, Universit
a degli Studi di Firenze, Viale G.B. Morgagni 85, 50134 Florence, Italy c Department of Bioscience and Biotechnology, Graduate School Kyushu University, Fukuoka, Japan

Received 24 February 2003, and in revised form 8 April 2003

Abstract Neutral ceramidase (CDase) is a key enzyme of sphingomyelin (SM) metabolism implicated in cell signaling triggered by a variety of extracellular ligands. Previously it was shown that in murine endothelial cells a portion of neutral CDase is localized in detergentresistant light membranes. In this study subcellular distribution of neutral CDase was further investigated. In accordance with the previous finding, the enzyme was identified in caveolae. Moreover, the same protein was detected in medium-speed supernatant of cell-conditioned medium, accounting for CDase activity measurable in the medium at neutral pH. Notably, these cells released also the caveolae-scaffolding protein caveolin-1 (cav-1). Interestingly, secreted neutral CDase and cav-1 coimmunoprecipitated. In addition, acid sphingomyelinase (SMase) activity was detectable in cav-1 immunocomplexes. These findings are consistent with the view that neutral CDase is released, in part, in association with cav-1 together with acid SMase. It remains to be established whether the here-identified secreted cav-1-enriched complex acts as platform to facilitate extracellular metabolism of SM. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Acid sphingomyelinase; Caveolin; Caveolae; Murine endothelial cells; Neutral ceramidase

Numerous studies performed over the past decade have emphasized the importance of sphingolipids and their metabolites as key components of the cell signaling machinery [1–3]. Ceramide, the precursor of all major sphingolipids in eukaryotes, is thought to serve as a second messenger for cellular functions ranging from differentiation to growth arrest and apoptosis [2,3]. In addition, more recently, evidence has been provided that ceramide plays an important role as membrane structural component, participating in receptor clustering and in vesicle formation and fusion [4]. Sphingosine, generated in vivo exclusively by N-deacylation of ceramide, catalyzed by ceramidase (CDase)1 [5], on its own can act either in a mitogenic [6,7] or a proapoptotic manner [8], but it can also serve as substrate for sphin-

gosine kinase (SphK) to yield sphingosine 1-phosphate (S1P), an intracellular and extracellular mediator, which acts as a powerful mitogen in various cell types [9]. In this context, CDase activity has to be regarded as the key regulator of the balance between ceramide and sphingosine cellular levels, responsible for the coordinated regulation of cell fate leading to proliferation or apoptosis. Multiple CDase isoforms differing in their catalytic pH optima have been identified so far in higher eukaryotes. Human lysosomal acid CDase was cloned first [10]; subsequent cloning of membrane-neutral CDase in mouse [11], human [12], and rat [13] further revealed the occurrence of distinct enzyme isoforms, possibly differing in subcellular localization and physiological roles. In

* Corresponding author. Fax: +39-055-422-2725. E-mail address: paola.bruni@unifi.it (P. Bruni). 1 Abbreviations used: CDase, ceramidase; S1P, sphingosine 1-phosphate; SMase, sphingomyelinase; SM, sphingomyelin; DRLM, detergent-resistant

light membranes; HDM, high-density membranes; cav-1, caveolin-1; TBS, Tris-buffered saline; TTBS, TBS with 0.1% Tween 20; Mes, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MBS, Mes-buffered saline; SphK, sphingosine kinase.

0003-9861/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00212-1

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this regard, human neutral CDase was identified in mitochondria when overexpressed in HEK 293 and MCF7 cells [12], whereas the enzyme was found mainly distributed in endosome-like structures in mouse liver and enriched in the raft microdomains of apical membranes in rat kidney [13]. Previously we focused our attention on the cellular localization of neutral CDase in the H.end cell line [14], which was originally isolated from murine hemangioma and immortalized by middle T antigen transformation and represents a suitable model to investigate endothelial cells since it retains functional properties of normal endothelial cells [15,16]. In these cells we detected neutral CDase in detergent-resistant light membranes (DRLM). Moreover these membrane microdomains also displayed acid and neutral SMase enzymatic activities [14], demonstrating that a specific compartmentalization of enzymes involved in the sphingomyelin (SM) cycle occurs in the plasma membrane. Cav-1, which serves as the major scaffolding protein of caveolae, is secreted by multiple cell systems including exocrine [17] and cancer cells [18]. Moreover, in an androgen-insensitive prostatic cancer cell line, released cav-1 has been demonstrated to bear biological activity, being capable of exerting a prometastatic action [18]. Given that CDase and SMase activities were found to be secreted by murine endothelial cells [19], in the present study subcellular distribution of neutral CDase and its release from murine endothelial cells were further investigated to address the question whether cav-1 acts as a scaffold for secreted SM-metabolizing enzymes. Here we report that a portion of neutral CDase localized at the plasma membrane was detectable in caveolae and that the secreted neutral CDase was in part associated to cav-1. Moreover, acid SMase activity was also measured in immunocomplexes of secreted cav-1, indicating the existence of a molecular platform for SM metabolism capable of acting in the extracellular milieu.

Materials and methods Materials H.end.FB. murine endothelial cells were a kind gift of Professor F. Bussolino (University of Turin, Italy). Reagents, materials for cell cultures, goat serum, protein A/G-Sepharose, and protease inhibitor cocktail were obtained from Sigma (St. Louis, MO, USA). BODIPY FL-SM and anti-rabbit IgG conjugated to Alexa Fluor 594 were obtained from Molecular Probes (Leiden, The Netherlands). C12-NBD-ceramide was prepared according to the method described [20]. Precoated Silica Gel 60 TLC plates were obtained from Merck (Darmstadt, Germany). Centrifugal filter units (molecular

weight cut-off of 10 kDa) were purchased from Millipore (Watford, England). I-block was from Tropix (Bedford, MA, USA). Vectashield mounting medium for fluorescence was from Vector Labs (Burlingame, CA, USA). [32 P]ATP (800 Ci/mmol) was purchased from NEN Life Science (Boston, MA, USA). Enhanced chemiluminescence reagents (ECL) were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Neutral recombinant mouse CDase was purified and utilized to generate rabbit polyclonal anti-CDase antibodies as described in [13]; in some experiments anti-CDase antibodies generated by Professor A. Huwiler [21] were utilized. Mouse monoclonal antibodies against cav-1 were from Transduction Laboratories (Lexington, KY, USA); rabbit polyclonal antibodies against cav-1 and horseradish peroxidase-conjugated anti-rabbit immunoglobulins were from Santa Cruz (Santa Cruz, CA, USA). Gold-conjugated anti-rabbit and anti-mouse immunoglobulins (10-nm gold particles) were from Calbiochem (Cambridge, MA, USA). Cell culture H.end.FB cells were grown in 100-mm dishes in DulbeccoÕs modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum and penicillin G (100 U/ml) plus streptomycin (100 lg/ml), maintained at 37 °C in a humidified atmosphere of 5% CO2 and utilized for experiments when 90% confluent. In some circumstances cells were shifted to serum-free medium and after 18 h conditioned medium was collected and spun 15 min at 20,000g. The supernatant was concentrated approximately 200-fold using a centrifugal filter unit. Preparation of cell extracts and sucrose density gradient cell fractionation Cells were washed twice with ice-cold PBS, scraped into Mes-buffered saline (MBS) (25 mM Mes, pH 6.5, 0.15 M NaCl, 1% Triton X-100) containing protease inhibitors, and homogenized with a Dounce homogenizer (100 strokes). Then cell lysates were subjected to sucrose density gradient centrifugation essentially as described [14]. Briefly, the homogenate adjusted to 40% sucrose was placed at the bottom of an ultracentrifuge tube and overlaid with two layers of 30 and 5% sucrose in MBS. The gradient was then centrifuged at 170,000g for 18 h using a Beckman SW50 rotor. Fractions of 0.4 ml were collected from the top of the gradient and used for protein content assay according to the Coomassie blue method. Immunoblot and immunoprecipitation analyses Protein aliquots were separated by SDS–PAGE and then electrotransferred to nitrocellulose membranes,

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which were incubated overnight in Tris-buffered saline containing 0.1% Tween 20 (TTBS) and 0.5% I-block. Hybridization for 1 h at room temperature with primary antibodies was followed by washing with TTBS and incubation with peroxidase-conjugated goat anti-mouse or anti-rabbit IgG. Proteins were detected by enhanced chemiluminescence. For immunoprecipitation analysis of released proteins, supernatant of culture medium of approximately 6
107 cells was concentrated 200-fold and incubated overnight at 4 °C with anti-cav-1 antibodies (1:100) or anti-neutral CDase antibodies (1:50) in buffer A (25 mM Tris, pH 7.4, 1% Triton X-100, 60 mM n-octyl-b-glucopyranoside, protease inhibitor cocktail). The immunocomplexes were collected after incubation for 2 h with protein A/G-Sepharose beads, washed twice with 25 mM Tris, pH 7.5, resuspended in 25 mM Tris, pH 7.5 (100 ll), and then used for Western analysis and enzyme activity determination.

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analysis using Imaging and Analysis Software by BioRad (Quantity-One). Indirect immunofluorescence H.end cells were grown on sterilized glass coverslips, fixed by incubation in 2% paraformaldehyde for 10 min, and blocked with 10% goat serum. The fixed cells were incubated overnight at 4 °C with anti-neutral CDase antibodies (1:100 dilution). After washing with PBS containing 1% BSA (PBS/BSA), bound primary antibodies were detected with anti-rabbit IgG conjugated to Alexa Fluor 594 (1:200 dilution). The coverslips were rinsed extensively with PBS/BSA and mounted in antifade Vectashield reagent. The immunofluorescence signal was visualized and photographed using a confocal Bio-Rad MRC 1024 ES scanning microscopy (Bio-Rad, Hercules, CA). Immunoelectron microscopy

Enzymatic assays CDase activity was determined using C12-NBD-ceramide as a substrate [19]. Briefly, 100 pmol of C12NBD-ceramide (NBD-C12:0, d18:1) were incubated 2 h at 37 °C with an appropriate amount of protein in 20 ll of 25 mM Tris–HCl buffer, pH 7.5, and 0.25% (w/v) Triton X-100. Samples were then applied to a TLC plate, which was developed with chloroform, methanol, 25% ammonia (90:20:0.5, v/v). Spots corresponding to NBD-dodecanoic acid and C12-NBD-ceramide were scraped, and incubated with methanol at 37 °C to extract the compounds from the silica, and their fluorescence at 470/525 nm excitation/emission wavelengths was measured using a Shimadzu 9000 spectrophotofluorimeter. The compounds were quantified using a standard curve of known amounts of C12-NBD-ceramide and NBD-dodecanoic acid. Acid SMase activity was measured at pH 5.0 utilizing a spectrophotofluorimetric assay based on detection of the rate by which BODIPY FL-ceramide was released from BODIPY FL-SM, essentially as described previously [22]. Assay of SphK activity was performed according to Ancellin et al. [23]. Briefly, aliquots of the medium-speed supernatant of culture medium (up to 150 lg protein) or cell lysates, used as control, were incubated in the presence of 20 lM sphingosine (dissolved in 0.5% Triton X-100) and 0.5 mM [32 P]ATP (10 lCi) in a buffer containing 15 mM NaF, 5 mM MgCl2 , 40 mM b-glycerophosphate, and 0.5 mM 4-deoxypyridoxine. Reactions were initiated by addition of [32 P]ATP and incubated for 2 h at 37 °C. Successively, lipids were extracted and separated by TLC with 1-butanol/acetic acid/water (3:1:1, v/v). Bands corresponding to S1P were visualized by autoradiography and quantified by densitometric

Cells were plated on cellulose membrane. After fixation in 2% paraformaldehyde, cells were stained with either polyclonal antibody against neutral CDase or monoclonal antibody against cav-1 diluted 1:100 in 50 mM Tris, pH 7.4, 150 mM NaCl containing 0.1% BSA (TBS), for 1 h at room temperature. After washing in TBS (pH 8.2), cells were subsequently incubated with anti-rabbit or anti-mouse IgG conjugated with 10-nm colloidal gold particles diluted in TBS (pH 8.2). Membranes with the adherent cells were then additionally fixed with 4% cold glutaraldehyde for 1 h, dehydrated, and embedded in Epon 812. Ultra-thin sections were taken from the embedded samples, collected on copper grids, and stained with uranyl acetate and alkaline bismuth subnitrate. The sections were then analyzed using a Jeol 1010 transmission electron microscope.

Results Immunofluorescence was utilized to assess cellular distribution of neutral CDase in H.end murine endothelial cells. As shown in Fig. 1A, most of the antineutral CDase staining was associated with cell plasma membrane, although a strong signal was detectable also in intracellular districts. Analysis of neutral CDase distribution performed by Western analysis confirmed that the ceramide-hydrolyzing activity was widely spread in membrane fractions. Indeed, antiserum against neutral CDase recognized a double band of 95 and 105 kDa in DRLM and in high-density membranes (HDM) prepared from H.end cells (Fig. 1B). Notably, neutral CDase was also detected as a double band in renal mesangial cells [21]. In the light of the recent identification of neutral CDase as glycoprotein [24], it is

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activity, in agreement with the localization in this fraction of the majority of cellular protein content [14], while only a minor fraction of cellular CDase was localized in DRLM. Since the identification of neutral CDase in DRLM does not necessarily imply that the protein is localized in caveolae, to further investigate this point immunoelectron microscopic analysis was performed. As shown in Fig. 1C, H.end cells exhibited classic flask-shaped membrane invaginations corresponding to caveolae. Notably, immunogold labeling of Epon 812-embedded samples with anti-neutral CDase polyclonal antibodies showed that caveolae were decorated with gold (arrows), demonstrating the specific localization of the enzyme in these membrane microdomains. According to its specific subcellular localization, cav1 was detected by immunoelectron microscopic analysis in caveolae of H.end cells (Fig. 2A) and in DRLM by immunoblotting experiments (Fig. 2B). Strikingly, as shown in the same figure, Western analysis of mediumspeed supernatant of conditioned medium displayed the

Fig. 1. Immunolocalization of neutral CDase in H.end cells. (A) Immunofluorescence staining. H.end cells were processed for immunofluorescence localization as described under Materials and methods and images visualized at confocal laser microscope. Neutral CDase was revealed using specific polyclonal anti-neutral CDase antibodies and anti-rabbit IgG conjugated to Alexa Fluor 594. Image presented is representative of four independent experiments. (B) Western analysis. H.end cells were fractionated in the presence of 1% Triton X-100 using a sucrose density gradient centrifugation to isolate DRLM. Cell medium was obtained concentrating the medium-speed supernatant of the conditioned medium of approximately 6
107 cells as described under Materials and methods. Samples (60 lg of protein from DRLM and 40 lg of protein from cell medium) were separated onto SDS–PAGE; neutral CDase was immunodetected using specific polyclonal antibodies. Recombinant mouse liver CDase was used as control. Neutral CDase activity was assayed as described under Materials and methods (n ¼ 4). A blot representative of three is reported. (C) Immunoelectron cytochemistry. Epon 812-embedded samples of H.end cells were processed for immunogold labeling of anti-neutral CDase polyclonal antibody (arrows). Final magnification 80,000
. The photographs presented are representative of three independent experiments.

conceivable that the two bands corresponded to proteins with different molecular mass consequent to variance in glycan composition, i.e., containing both complex-type N-glycans and O-glycans or only high-mannose type Nglycans. Neutral CDase was immunorevealed in the supernatant of conditioned medium and in DRLM and HDM of murine endothelial cells (Fig. 1B). Moreover, as shown in the same figure, ceramide-hydrolyzing activity could also be detected in all the samples: HDM contained a high percentage of the whole-cell enzymatic

Fig. 2. Immunolocalization of cav-1 in H.end cells. (A) Immunoelectron cytochemistry. Immunogold labeling of mouse monoclonal anticav-1 antibodies in H.end cells was performed as described under Materials and methods. Final magnification was 100,000
. The photographs presented are representative of three independent experiments. (B) Western analysis. Samples obtained from DRLM or from cell medium (0.6 lg or 40 lg of protein, respectively) were separated onto SDS–PAGE and electrotransferred to nitrocellulose membranes. Cav-1 was detected using specific rabbit polyclonal anti-cav-1 antibodies. A blot representative of three is shown.

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presence of a 21-kDa band corresponding to cav-1. However, the higher amount of protein required to detect cav-1 in the conditioned medium (40 lg versus 0.6 lg) demonstrated that cav-1 was less represented in the extracellular milieu in comparison with DRLM. These results indicate that cav-1, localized in the abundant caveolae of H.end cells, is also discharged into the cell medium. Secretion of cav-1 and neutral CDase by H.end murine endothelial cells was further investigated by analyzing protein immunocomplexes. Notably, cav-1 and neutral CDase were specifically coimmunoprecipitated from the medium-speed supernatant of cell-conditioned medium (Fig. 3), consistent with the view that neutral CDase is released into the medium, in part, associated with cav-1. Moreover, cav-1 immunocomplex

Fig. 3. Coimmunoprecipitation of neutral CDase and cav-1 in cell medium of H.end cells. Samples (400 lg of protein) from concentrated cell medium were either immunoprecipitated (IP) with (+) or without ()) anti-mouse cav-1 antibodies (1:100) and revealed with an antineutral CDase immunoblot (w) or immunoprecipitated (IP) with (+) or without ()) anti-neutral CDase antibodies (1:50) and revealed with anti-cav-1 immunoblot (w). The experiment was repeated three times with analogous results.

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displayed neutral CDase activity (30  2.3 pmol/h (n ¼ 3) in the immunoprecipitate from 400 lg protein of conditioned medium), demonstrating that the secreted enzyme associated to cav-1 was catalytically active. However, the relatively low activity measured in cav-1 immunocomplexes, in comparison with that exhibited by the conditioned medium (Fig. 1B), suggested that neutral CDase associated to cav-1 represented only a fraction of the total secreted enzyme. Additionally, acid SMase activity could also be detected in cav-1 immunoprecipitates (210  25 pmol/h (n ¼ 3) in the immunocomplex from 400 lg protein of medium-speed supernatant of conditioned medium), consistent with the hypothesis that cav-1 was secreted as a complex bearing two main enzymatic activities involved in SM metabolism. Indeed, immunolocalization of neutral CDase by preembedding immunoelectron microscopy performed in cells with normal ultrastructural features (Figs. 4A and C) shows that the protein was localized at budding areas of the plasma membrane (Fig. 4B), likely involved in the release of membrane components. Similarly, cav-1 was found in analogous cytoplasmic projections (Fig. 4D), suggesting the existence of a common pathway for the two proteins leading to their discharge into the medium. In view of the recent identification of SphK as an enzymatic activity exported by the endothelial cells in the extracellular milieu and possibly involved in the extracellular biosynthesis of S1P, the medium-speed supernatant of the cell-conditioned medium was tested for the presence of SphK. No appreciable formation of labeled S1P could be measured, using up to 150 lg protein in the assay, although SphK was detectable in 10 lg of crude cellular extract (data not shown).

Fig. 4. Immunocytochemical localization of CDase and cav-1 in plasma membrane projections of H.end cells. H.end cells were fixed and embedded in Epon 812. Immunostained cells with normal ultrastructure are shown (A,C) (final magnification 4200
and 5000
, respectively) Immunogold labeling using monoclonal antibodies against neutral CDase (B) (final magnification 45,000
) or polyclonal antibodies against cav-1 (D) (final magnification 65,000
) was done as described under Materials and methods. Numerous colloidal gold particles (10-nm gold) recognizing both antigens are visible along the cell surfaces and over apical cytoplasmic projections (arrowheads). The photographs presented are representative of three independent experiments.

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Discussion Neutral CDase appears to be a key enzyme of SM metabolism and is believed to be implicated in cell signaling of a variety of ligands [3]. Recent studies have reported the occurrence of neutral CDase in various subcellular compartments, such as endosomes, raft microdomains, and mitochondria, suggesting that intracellular localization of the enzyme may be cell specific [11–13]. In a previous study we showed that in H.end murine endothelial cells neutral CDase was localized in membranes and was detectable in DRLM [14]. Here, we further characterized subcellular localization of neutral CDase in endothelial cells. Immunofluorescence and immunoelectron cytochemistry demonstrated that neutral CDase was localized at plasma membrane in caveolae, in noncaveolar districts, and in other intracellular compartments. Notably, this finding is particularly interesting in view of a recent study showing that ceramide reorganization of lipid rafts is essential for cellular response and defense against acute Pseudomonas aeruginosa infection [25]. Indeed, CDase localized in these microdomains could play a role in platform dynamics. Moreover, here, we provide experimental evidence that this enzyme is secreted by endothelial cells and that a portion of the protein exported to the extracellular milieu is associated to cav-1. Indeed, neutral CDase was detectable by immunoblotting of cav-1 immunocomplex and cav-1 was revealed in neutral CDase immunocomplexes. Furthermore, immunoelectron cytochemistry identified cav1 and neutral CDase in budding areas of the plasma membrane, suggesting that they could be released by the discharge of vesicles. These data confirm that cav-1 can be released by various cell types [17,18]. Interestingly, this is the first evidence for the association of CDase to cav-1. Another finding of the present study is the identification of acid SMase activity in cav-1 immunocomplexes. Strikingly, this enzymatic activity is known to be discharged into the medium by murine endothelial cells [19] and colocalizes with neutral CDase in DRLM [14]; these findings demonstrate that acid SMase and neutral CDase are subjected to a common intracellular processing leading to their combined export from cells in association to cav-1. Our data indicate that only a fraction of the total secreted neutral CDase is recovered in association with cav-1, suggesting that different pools of the enzyme contribute to the export of the neutral CDase to the extracellular milieu. In this regard, a mucin-like domain in neutral CDase that must be O-glycosylated to retain the enzyme on the plasma membrane as a type II integral membrane protein has been recently identified. It was also found that the domain was occasionally lost by posttranslational processing, resulting in a different

localization of the enzyme, i.e., release into the extracellular milieu [24]. In the light of these findings, it is possible to hypothesize that a significant pool of secreted neutral CDase is represented by the soluble form of the enzyme, presumably lacking the mucin-like domain and constitutively discharged into the medium. In addition, a second minor pool of the secreted enzyme would be represented by the neutral CDase localized in caveolae as integral membrane protein and discharged into the medium associated with cav-1 and acid SMase in the shape of vesicles. Intriguingly, several proteins localized in caveolae or lipid rafts, such as CD95 [26], matrix metalloproteinase-1 and -2 [27,28], and urokinase receptor [29], resulted in being discharged into medium [30–32], suggesting that such a membrane microdomain localization can be critical for secretion. Moreover, given that we previously noticed that bradykinin or phorbol esters stimulate CDase secretion by H.end cells [19], it is possible that the amount of secreted CDase associated with cav-1 is regulated by extracellular cues. An intriguing point that is worthy of future investigation is represented by the possible physiological function exerted by secreted CDase and SMase activities associated to cav-1. In view of the recently proposed role for secreted SMase and CDase activities, together with SphK, in the extracellular biosynthesis of S1P [33], it is tempting to speculate that the release of cav-1-enriched complexes bearing CDase and SMase would greatly enhance the efficiency of extracellular SM metabolism toward S1P formation. Indeed, even though in this study it was not possible to show appreciable secretion of SphK, it is plausible that the release of the enzyme can take place in response to not yet identified cell agonists. Finally, it has been reported that cav-1 released by prostate cancer cells is capable of exerting a prometastatic action [18]. However, in that study whether secreted cav-1 was associated to enzymes that could account for the observed biological activity was not examined. On the other hand, cav-1 was identified in tumor cell membrane-shed vesicles, enriched in gangliosides [34], but no further information on possible colocalization of the protein with enzymatic activities was provided. The present observed association of cav-1 to CDase and SMase activities leads to the hypothesise that these enzymes could be responsible, at least in part, for the biological activity exerted by secreted cav-1. A better knowledge of the context in which secreted cav-1/enzyme complex acts should expand our understanding of the overall biological role of cav-1.

Acknowledgments This work was supported by funds from the Cofinanziamento Ministero dellÕUniversit
a e della Ricerca

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