RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44

Nathalie Charnaux1,3, Séverine Brule1,3, Thomas Chaigneau3, Line Saffar3, Angela Sutton3, Morgan Hamon3, Catherine Prost3, Nicole Lievre3, Claudio Vita4 and Liliane Gattegno2,3

3 Laboratoire de Biologie Cellulaire, Biothérapies Bénéfices et Risques, UPRES 3410 Université Paris XIII, 74, rue Marcel Cachin, 93017, Bobigny, France, Hôpital Jean Verdier, 93017, Bondy; and 4 CEA, Saclay, Département d'Ingénierie et d'Etudes des Protéines, 91191 Gif-sur-Yvette, France


2 To whom correspondence should be addressed; e-mail: liliane.gattegno{at}jvr.ap-hop-paris.fr

Received on June 8, 2004; revised on August 3, 2004; accepted on September 3, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase–and protein kinase C–dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.

Key words: chemokine / proteoglycan / RANTES / syndecan-1 / syndecan-4


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Chemokines play critical roles in leukocyte recruitment into sites of inflammation (Luster, 1998Go; Zlotnik and Yoshie, 2000Go). Heparan sulfate (HS) is known to mediate cell surface oligomerization of chemokines. This indicates an HS-induced activation of the chemokines (Hoogewerf et al., 1997Go; Kuschert et al., 1999Go). Much of the HS at the cell surface is derived from the syndecan (SD) family of transmembrane proteoglycan (PG) (Bernfield et al., 1992Go). The SDs bind a variety of growth factors, cytokines, proteases, antiproteases, and cell adhesion molecules (Bernfield et al., 1999Go; Zimmermann and David, 1999Go); they are individually expressed in distinct cell-, tissue-, and development-specific patterns (Kim et al., 1994Go) and show cell-specific variations in the structure of their HS chains (Kato et al., 1994Go, Sanderson et al., 1994Go). CD44 is a multistructural and multifunctional cell adhesion molecule that is involved in cell–cell and cell–matrix interactions (Naot et al., 1997Go; Ponta et al., 2003Go).

SD-1 (CD138) is the best characterized of the four SD core proteins, binding to a variety of components of the extracellular matrix (Couchman et al., 2001Go; Woods and Couchman, 1998Go). SDs may regulate ligand-dependent activation of cell surface growth factor receptors by several potential mechanisms, including sequestering and increasing the effective concentrations of growth factor, increasing the range of binding interactions with ligands at the cell surface, or sequestering bound ligands with specific plasma domains (Bernfield et al., 1999Go; Clasper et al., 1999Go; Zimmermann and David, 1999Go). We have recently shown that regulated on activation normal T cell expressed and secreted (RANTES) forms multimolecular complexes on the plasma membrane of CCR5-positive HeLa cells and human primary macrophages. These complexes make up the PGs SD-1 and SD-4 (Slimani et al., 2003aGo,bGo), beside CCR5 and CD44 (Roscic-Mrkic et al., 2003Go, unpublished data). Moreover, we have also recently suggested by the use of coimmunoprecipitation experiments the occurrence of an heteromeric complex on the plasma membrane of these cells, which makes up CCR5, SD-1, and SD-4 but neither the PGs, CD44, nor betaglycan (Slimani et al., 2003aGo, unpublished data). These data argue for different physiological roles of the different RANTES-bound PGs. Moreover, RANTES also forms complexes with SD-1, SD-4, and CD44 on the plasma membrane of HeLa cells lacking CCR5. Therefore, even in the absence of its G protein-coupled receptors (GPCRs), RANTES binds PGs (Slimani et al., 2003aGo, unpublished data).

The ectodomain of each SD is constitutively shed from cultured cells. Ectodomain shedding appears to contribute to diverse pathophysiological events (Bernfield et al., 1999Go). It is therefore known that SD-1 and SD-4 are constitutively shed by cultured cells (Jalkanen et al., 1987Go) and that this shedding involves release of the soluble ectodomains (Saunders et al., 1989Go). Moreover, CD44 shedding by proteolytic processing was also reported (Brennan et al., 1999Go; Cichy and Pure, 2004Go). The site of cleavage has been suggested to be a dibasic sequence for SD-1, or a basic residue for SD-4, adjacent to the plasma membrane (Bernfield et al., 1992Go; Jalkanen et al., 1987Go). SD-1 and SD-4 shedding is accelerated within minutes of treating cells with the protein kinase C (PKC) activator, phorbol 12-myristate13-acetate (PMA) (Fitzgerald et al., 2000Go; Subramanian et al., 1997Go). However, how shedding is regulated remains largely unknown.

The present study was undertaken to investigate whether RANTES directly binds to the PGs SD-1 (CD138), SD-4, and CD44 expressed by HeLa cells as well as to their shed ectodomains and whether this chemokine accelerates the shedding of these PGs by HeLa cells expressing or lacking CCR5 and forms complexes with shed ectodomains of these PGs.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Binding of RANTES to SD-1, SD-4, and CD44 expressed by HeLa cells
We have previously shown by coimmunoprecipitation that RANTES forms complexes with CCR5 and two PGs belonging to the SD family, SD-1 and SD-4, on the plasma membrane of CCR5-positive HeLa cells or of monocyte-derived macrophages (Slimani et al., 2003aGo). However, complexes including RANTES, SD-1, and SD-4 can also be formed if the HeLa cells are CCR5-negative (Slimani et al., 2003bGo). Moreover, others have demonstrated that RANTES can be coimmunoprecipitated with CD44 expressed by HeLa cells (Roscic-Mrkic et al., 2003Go).

The purpose of our first experiments was to provide evidence for the occurrence of a direct binding of RANTES to SD-1 and SD-4 and also to CD44. The anti-SD-1, anti-SD-4, and anti-CD44 monoclonal antibodies used here have already been characterized (Gallo et al., 1996Go; Hamon et al., 2004Go; Kainulainen et al., 1998Go; Slimani et al., 2003bGo). Both HeLa cell lines used here express CXCR4, SD-2, betaglycan (data not shown) (Chang et al., 2002Go; Hamon et al., 2004Go), high levels of CD44, and relatively lower levels of SD-1 and SD-4 as assessed by flow cytometry analysis after indirect immunofluorescence labeling (Figure 1A). The HeLa P4 cells express in addition CCR5 and CD4 (data not shown) (Slimani et al., 2003bGo). The PGs-enriched fraction obtained from lysates of CCR5-positive or -negative HeLa cells were further characterized (Hamon et al., 2004Go) after treatment by heparitinase I, III, and chondroitinase ABC mixture. In such conditions, 32-kDa and 50–58-kDa proteins, immunoreactive with both anti-SD-4 5G9 and 3G10 monoclonal antibodies were observed (Figure 1B and data not shown). The 50–58-kDa proteins may represent homo- or hetero-oligomers of the SD4-core protein, which is a 32-kDa protein (Oh et al., 1997Go). Other proteins were also detected: 34-kDa proteins immunoreactive with anti-SD-2 antibody and monoclonal antibody 3G10, 45- and 90-kDa proteins immunoreactive with anti-SD-1 DL-101 and 3G10 antibodies, 90-kDa proteins likely dimers of the 45-kDa ones, and 60-kDa proteins immunoreactive with anti-CD44 antibody and 3G10 (Figure 1B and data not shown). Because antibody 3G10 reacts with an epitope including a terminal unsaturated uronic acid residue that is unmasked after HS removal by heparitinase treatment (David et al., 1992Go), these data demonstrate that all the PGs characterized in these experiments were glycanated before their glycosaminidase treatment. Moreover, the apparent molecular masses of these PGs core proteins are close to the predicted ones (Bernfield et al., 1992Go; 1999Go).



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Fig. 1. Expression of proteoglycans on HeLa cells. (A) Cell surface expression of SD-1, SD-4, and CD44 on HeLa cells. 5 x 105 CCR5-positive HeLa cells were stained for FACS analysis with anti-SD-1 DL-101 (a), anti-syndecan-4 5G9 (b) or anti-CD44 (c) monoclonal antibodies (thick lines). Reactivity was compared to an isotype-matched control monoclonal Ab (a, b, c, dotted lines). Data are representative of three individual experiments. (B) Immunoblot analysis of proteoglycans from HeLa cells. HeLa cells were lysed in the presence of Triton X-100 and urea. PGs (from 2 x 106 cells per lane) were enriched by DEAE Sephacel anion exchange chromatography and then treated with heparitinases I, III, and chondroitinase ABC mixture, electroblotted, and revealed with 3G10 monoclonal antibody (lane 1) or the isotype, IgG2b (lane 2). The respective immunoreactivity with anti-SD-1 DL-101, anti-SD-4 5G9, anti-CD44 antibodies, and anti-SD-2 antibodies are represented by arrows. Data are representative of three individual experiments.

 
We then investigated whether RANTES directly binds to some of the glycanated PGs previously enriched from the HeLa cells lysates by DEAE Sephacel anion exchange chromatography. We observed that electroblotted glycanated SD-1 migrated as a 98-kDa band immunoreactive with anti-SD-1 antibody DL-101, glycanated SD-4 as a 200–250-kDa smear immunoreactive with anti-SD-4 antibody 5G9, and glycanated CD44 as a 110-kDa band. No immunoreactivity was detected with the isotype-matched control antibodies (Figure 2, lanes 1–5). The fact that all these bands were immunoreactive with anti-HS antibody 10E4 but not with its isotype further indicates that these PGs are glycanated (Figure 2, lanes 6, 8).



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Fig. 2. Biotinylated-RANTES binds SD-1, SD-4, and CD44. HeLa cells were lysed in the presence of Triton X-100 and urea. PGs were enriched by DEAE Sephacel anion exchange chromatography, electroblotted, and revealed with anti-SD-1 DL-101 (lane 1), anti-SD-4 5G9 (lane 3), anti-CD44 (lane 5), anti-HS 10E4 (lanes 6) monoclonal antibodies and in parallel with biotinylated RANTES (lanes 9) or the respective isotypes IgG1 (lane 2), IgG2a (lane 4), IgM (lane 8), or with a streptavidin-peroxidase complex (lane 12). Alternatively, strips were treated with heparitinases I, III mixture and revealed with 10E4 antibody (lane 7), biotinylated RANTES (lane 10), anti-SD DL-101 (lane 13), anti-SD-4 5G9 (lane 14), or anti-CD44 (lane 15) antibodies. In some experiments, biotinylated RANTES was preincubated with 330 µg/ml of heparin and then incubated with the strips (lane 11). Data are representative of three individual experiments.

 
Biotinylated RANTES bound to electroblotted glycanated SD-1, SD-4, and CD44 (Figure 2, lane 9). Glycosaminidase (heparitinases I and III, chondroitinase ABC) pretreatment of the electroblotted PGs was efficient because it abolished the binding of these PGs to anti-HS antibody 10E4 (Figure 2, lane 7). However, the fact that after this enzyme treatment, the respective immunoreactivity of the PGs toward their respective monoclonal antibody was not decreased, indicates that the PGs are still present on the membrane (Figure 2, lanes 13–15). Nevertheless, this pretreatment abolished the binding of biotinylated RANTES to the respective electroblotted PGs, SD-1, SD-4, and CD44 (Figure 2, lane 10). These data demonstrate the direct and glycosaminoglycan (GAG)-dependent binding of RANTES to SD-1, SD-4, and CD44. Because preincubation of RANTES with heparin inhibits these bindings, this GAG may act as a competitive inhibitor (Figure 2, lane 11).

RANTES accelerates the shedding of SD-1 and SD-4 from CCR5-positive HeLa cells
To investigate whether RANTES accelerates the shedding of the ectodomains of its interacting PGs, we first studied whether SD-1, SD-4, and CD44 are constitutively shed from HeLa cells. As positive control, we analyzed the effects of PMA on these sheddings. Chemokine domains and residues that are important for the GPCR binding and/or activation, have been identified (Blanpain et al., 1999Go; Samson et al., 1997Go). How some chemokines interact and activate GPCRs and other cell membrane ligands is still not fully elucidated. It has recently been shown that the core domain of RANTES binds CCR5 extracellular domains, whereas its amino terminus interacts with the transmembrane helix bundle (Blanpain et al., 2003Go). Therefore, we investigated the effects of RANTES (1–68) or lacking the nine N-terminus amino-acids (10–68) on the shedding of SD-1 and SD-4 ectodomains.

We first showed the presence of proteins, respectively immunoreactive with anti-SD-1 BB4, anti-SD-4 5G9, and anti-CD44 monoclonal antibodies, in the culture supernatants of unstimulated CCR5-positive or -negative HeLa cells (Figure 3A). Such immunoreactive proteins were not detected if the culture medium was not previously incubated with the cells (data not shown). This indicates that SD-1, SD-4, and CD44 are constitutively shed from the HeLa cells whether they express CCR5 or not (Figure 3A, B, and data not shown).



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Fig. 3. RANTES accelerates the shedding of SD-1 and SD-4 from CCR5-positive HeLa cells. (A) CCR5-positive HeLa cells (CCR5+) or CCR5-negative HeLa cells (CCR5–) were incubated or not (untreated) for 16 h with PMA (0.5 µM) or RANTES (at 5 nM or 125 nM). Conditioned media were harvested and proteoglycans, partially purified by application to cationic membranes, were analyzed by dot blot. SD-1, SD-4, and CD44 were detected by ECL detection, using respectively anti-SD-1 BB4, anti-SD-4 5G9, or anti-CD44 antibodies. One of three individual experiments is shown. (B) The data are expressed as the amount of SD shed in absorbance units, measured by densitometric scanning and analyzed with an image software. Each point represents the mean±SE of triplicate determinations. One of three individual experiments is shown.

 
The shedding of SD-1 and SD-4 from these two cell lines was significantly increased (p < 0.01 and p < 0.05, respectively; three independent experiments) if the cells were incubated for 18 h at 37°C with 0.5 µM PMA, a PKC activator (Fitzgerald et al., 2000Go) (Figure 3A, B, and data not shown). However, PMA had no significant effect on the constitutive shedding of CD44 (p = 0.2, n = 3) (Figure 3A). PMA also significantly decreased SD-1 and SD-4 expression on the plasma membrane of these cells (p < 0.05 and p < 0.02, respectively, n = 3) (Figure 4 and data not shown), whereas it significantly increased CD44 expression (p < 0.05, n = 3), as assessed by cytofluorimetry analysis after indirect immunofluorescence labeling of the cells (Figure 4 and data not shown). The fact that PMA accelerates the shedding of SD-1 and SD-4 from HeLa cells expressing or lacking CCR5 suggests that such stimulations do not depend on CCR5.



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Fig. 4. Effect of incubating CCR5-positive HeLa cells with RANTES on SD-1, SD-4, and CD44 cell surface expression. CCR5-positive HeLa cells were incubated or not (untreated) for 16 h with PMA (0.5 µM) or RANTES 1–68 (125 nM) and then stained for FACS analysis with anti-SD-1 DL-101 (A), anti-SD-4 5G9 (B) or anti-CD44 antibodies (C). Reactivity was compared to an isotype-matched control monoclonal antibody. SD-1 (A), SD-4 (B), and CD44 (C) expressions were the mean percent of changes in mean fluorescence intensity (mean ± SE) as compared with that of unstimulated control cells. Each point represents the mean ± SE of triplicate determinations. One of three individual experiments is shown.

 
Interestingly, incubating RANTES (5 nM to 125 nM) for 18 h at 37°C with the CCR5-positive HeLa cells significantly increased the respective shedding of SD-1 and SD-4 (p < 0.05, n = 3), as compared with the data observed in RANTES- or PMA-free medium (Figure 3A, B); these increases slightly depended from RANTES concentration (Figure 3A). These accelerated sheddings were observed even if the cells were incubated with low but physiologically relevant RANTES concentrations (5 nM) (Figure 3A) or with 125 nM RANTES for 2 h (instead of 18 h) at 37°C (p < 0.05, n = 3) (Figure 5). However, RANTES did not change the constitutive shedding of CD44 from these cells (p = 0.5, n = 3) (Figure 3A). In addition, if synthetic RANTES 10–68 (125 nM) was incubated for 18 h at 37°C with the HeLa cells expressing CCR5, a significant increase of SD-1 and SD-4 sheddings was also observed (p < 0.05, n = 4) (data not shown). Finally, the plasma membrane expression of SD-1, and to a lower extent of SD-4, was significantlty decreased when the CCR5-positive HeLa cells were incubated with either RANTES (1–68) or RANTES (10–68) (respectively, p < 0.05 and p < 0.02, n = 3); by contrast, the expression of CD44 was significantly increased in these conditions (p < 0.05, n = 3) (Figure 4 and data not shown). Therefore, RANTES, at low physiological as well as high pathological concentrations (125 nM), induces an accelerated shedding of SD-1 and SD-4, but not of CD44, from HeLa cells expressing CCR5; the nine N-terminus amino acids of RANTES are not required for these accelerating effects. However, incubation of RANTES (125 nM, for 18 h at 37°C) with the CCR5-negative HeLa cells did not significantly accelerate the constitutive sheddings of SD-1 and SD-4 (respectively, p = 0.5 and 0.7, n = 3) (Figure 3A, B). Moreover, no changes in SD-1 and SD-4 membrane expression of these cells were observed (data not shown). In addition, RANTES changed neither the CD44 constitutive shedding from these cells (p = 0.5, n = 3) (Figure 3A) nor their CD44 plasma membrane expression (data not shown).



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Fig. 5. Inhibition of MAPK and PKC activities prevents the shedding of SD-1 and SD-4 accelerated by RANTES. (A) Serum-starved CCR5-positive HeLa cells were preincubated (lane 3) or not (lanes 1, 2) with the MEK1/2 inhibitor U0126 (10 µM) for 2 h and then stimulated (lanes 2, 3) or not (lane 1) for 15 min with RANTES (at 125 nM) and lysed as described in Materials and methods. Protein samples were run on SDS–PAGE (10%), electroblotted, and revealed with an antiphospho-p44/p42 antibody. Protein loading was controlled by using a total anti-p44/p42 antibody. One of three individual experiments is shown. (B) CCR5-positive HeLa cells were incubated for 2 h with or without (untreated) PMA (0.5 µM) (as control) or RANTES (125 nM). Each agonist was assayed in the presence or absence of the MEK1/2 inhibitor U0126 (10 µM) or of the PKC inhibitor bisindolylmaleimide I (Bis.) (1 µM). Conditioned media were analyzed by dot blots, which were revealed with anti-SD-1 BB4 (SD-1) or anti-SD-4 5G9 antibodies (SD-4). (C) The data are expressed as the amount of SD shed in absorbance units, measured by densitometric scanning and analyzed with an image software. Each point represents the mean±SE of triplicate determinations. One of three individual experiments is shown.

 
These data demonstrate that the presence of both CCR5 expressed on the plasma membrane and exogeneous RANTES (either 1–68 or 10–68) are necessary to significantly accelerate the sheddings of SD-1 and SD-4 from CCR5-positive HeLa cells and increase their CD44 expression. Although the accelerating effect on SD-1 and SD-4 shedding induced by RANTES depends on CCR5, that induced by PMA is CCR5-independent. This strongly suggests that different agonists, such as PMA or RANTES, may act on distinct intracellular signaling pathways to accelerate SD-1 and SD-4 shedding.

The G-protein-coupled pathway activated by RANTES through chemokine receptors involves an increased tyrosine phosphorylation, an activation of focal adhesion kinase and phospholipase D, a phosphorylation of ERK (p44/p42 MAPK), a member of the mitogen-actived protein kinase (MAPK) family (Bacon et al., 1996Go, 1998Go; Schwabe et al., 2003Go; Tilton et al., 2000Go), and also a protein kinase–mediated phosphorylation of four distinct C-terminal serine residues of CCR5 (Pollok-Kopp et al., 2003Go).

The compound U0126 inhibits two MAPKs, MEK-1 and MEK-2 (Favata et al., 1998Go); this inhibition is selective because U0126 has little, if any, effect on the kinase activities of PKC and other kinases (Favata et al., 1998Go). Moreover, signaling through GPCRs can activate the p44/42 MAPKs through both G alpha-dependent (Ptx-sensitive) and G alpha-independent (Ptx-insensitive) mechanisms (Luttrell et al., 1999Go). In the present study, we observe in accordance with others (Schwabe et al., 2003Go), that RANTES activates the phosphorylation of a member of the MAPK family: Indeed, incubation of the CCR5-positive HeLa cells with 125 nM RANTES induces the phosphorylation of the p44/p42 MAP kinases (Figure 5A) and addition of 10 µM U0126 to the incubation medium inhibits this enhanced phosphorylation (Figure 5A). In parallel, preincubation of the CCR5-positive HeLa cells for 2 h at 37°C with both RANTES (125 nM) and MEK 1/2 inhibitor U0126 (10 µM), also significantly prevented the shedding of SD-1 and SD-4 accelerated by RANTES (p < 0.05, n = 3) (Figure 5B, C). These data suggest that MEK-1 or MEK-2 are involved in these CCR5-dependent accelerated sheddings on CCR5-positive HeLa cells. However, in the same conditions, U0126 had no effect on the shedding of SD-1 and SD-4 accelerated by PMA (p = 0.2, n = 3) (Figure 5B, C).

We then asked whether the shedding of SD-1 and SD-4 accelerated by RANTES also involves PKC activity. For this purpose, we tested whether bisindolylmaleimide I, a potent and selective inhibitor of this pathway (Toullec et al., 1991Go), affected these sheddings. Although bisindolylmaleimide I prevented in a significant manner the sheddings of SD-1 and SD-4 accelerated by PMA, as expected (Fitzgerald et al., 2000Go), this compound also significantly prevented the shedding of SD-1 and SD-4 accelerated by RANTES (p < 0.01, n = 3) (Figure 5B, C). In each assay, we tested the effect of the MAPK or PKC inhibitor alone. In no case did the inhibitor affect the level of shedding compared with the untreated control (data not shown).

Taken together, these data suggest that the respective shedding of SD-1 and SD-4 accelerated by RANTES depends on both MEK1/2- and PKC-transduction pathways and rule out the involvement of MEK1/2 in the accelerated shedding of SD-1 and SD-4 induced by PMA.

RANTES induces an increase in SD-1 and SD-4 mRNA amounts
We next investigated whether SD-1 and SD-4 mRNA levels were induced by RANTES. For this purpose, we analyzed these PGs' mRNA levels in HeLa cells under different culture conditions. The SD-1 and SD-4 mRNA were both increased if the cells were incubated with PMA (p < 0.001, n = 3) or with RANTES (125 nM for 18 h at 37°C), whether the cells express or lack CCR5 (p < 0.001 and p < 0.01, n = 3) (Figure 6A, B). These data indicate that RANTES stimulates SD-1 mRNA and SD-4 mRNA synthesis in CCR5-negative as well as in CCR5-positive HeLa cells. However, the effects of such stimulation on the expression of these PGs and on their shedding, vary according to the presence of CCR5.



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Fig. 6. Effect of PMA or RANTES on SD-1 or SD-4 mRNA synthesis in CCR5-positive (A) or CCR5-negative (B) HeLa cells. (A) The effect of PMA or RANTES on SD-1 or SD-4 mRNA synthesis was studied by RT-PCR. PCR products were analyzed on agarose gel stained with ethidium bromide. Primers and conditions are specified in Materials and methods. PCR for the housekeeping gene GAPDH was used as a control for equal loading of RNA into the RT-PCR reaction tubes. Obtaining bands of consistent intensity for GAPDH allowed comparison of the amount of PCR products between different samples. The size of the PCR products were 211 bp for SD-1, 531 bp for SD-4, and 983 bp for GAPDH. (B) SD-1 or SD-4 mRNA levels were quantified by densitometric scanning and analyzed with an image software. Results are expressed as SD-1 or SD-4 mRNA level relative to the level of GAPDH mRNA. Each point represents the mean±SE of triplicate determinations. One of three individual experiments is shown. P of the coupled difference as compared to the corresponding untreated cells. * < 0.001; ** < 0.01.

 
RANTES forms complexes with the ectodomains of SD-1, SD-4, and CD44
We then investigated whether RANTES forms complexes with the constitutively shed ectodomains of SD-1, SD-4, and CD44, either present in the culture supernatant of the CCR5- positive HeLa cells, or in their enriched preparations. In both cases, after incubation for 18 h with RANTES (50–125 nM), then with anti-RANTES coated protein-G beads, the RANTES-interacting material was characterized as proteins, respectively, immunoreactive with anti-SD-1 DL-101, anti-SD-4 5G9, and anti-CD44 antibodies but not with the respective isotype-matched control antibodies (data not shown). No immunoreactivity was detected if the beads were coated with the isotype instead of the anti-RANTES antibody (data not shown).

We also studied whether RANTES forms complexes with the PGs if the CCR5-positive HeLa cells were stimulated for 18 h in the presence of 125 nM RANTES. In such conditions, the immunoprecipitated proteins show evidence of 40- and 50-kDa proteins, immunoreactive with anti-SD-1 antibody DL-101; 45-kDa proteins, immunoreactive with anti-SD-4 5G9; and 70-kDa proteins, immunoreactive with anti-CD44 antibody; no immunoreactivity with the respective isotypes was observed (Figure 7A). Similar apparent molecular masses of the shed ectodomains of these PGs were also observed in the culture supernatant (data not shown). The differences in the apparent molecular masses of the shed ectodomains of SD-1, SD-4, and CD-44 complexed with RANTES as compared to those of the intact PGs present in the cell lysates may be related to the small portion of the transmembrane and cytoplasmic domains of the PGs and/or to homo- or hetero-oligomerization process.



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Fig. 7. RANTES forms complexes with the shed ectodomains of SD-1, SD-4, and CD44. CCR5-positive HeLa cells were incubated for 18 h with RANTES (at 125 nM). (A) The conditioned supernatant medium was immunoprecipitated with anti-RANTES coated beads. Collected immunocomplexes were electroblotted and revealed with anti-SD-1 DL-101 (lane 1), anti-SD-4 5G9 (lane 2), anti-CD44 (lane 3) antibodies or their respective isotypes (lanes 4, 5). One of three individual experiments is shown. In these experiments, the immunoprecipitated proteins remained untreated (not digested with heparitinases). (B) The conditioned supernatant medium was immunoprecipitated with anti-SD-1 antibody DL-101 (lane 1), anti-SD-4 antibody 5G9 (lane 2), or anti-CD44 antibody (lane 3). Collected immunocomplexes were electroblotted and revealed with anti-RANTES monoclonal antibody (lanes 1–3) ot its isotype IgG1 (lanes 4–6). In lane 7, RANTES (5 µg), directly loaded for gel analysis, was used as control [c].

 
These data demonstrate that RANTES forms complexes with the shed ectodomains of SD-1, SD-4, and CD44. If the immunoprecipitated proteins described were respectively performed with anti-SD-1 DL-101, anti-SD-4 5G9, or anti-CD44 antibody-coated beads, RANTES coimmunoprecipitated with the PGs, as assessed by electroblotting and revelation with anti-RANTES antibody (Figure 7B). In the same conditions, no binding of SDF-1 (CXCL12), another chemokine, to the shed ectodomains of SD-1, SD-4, and CD44 was observed (data not shown). This may be related to the fact that although SDF-1 binds to PGs such as SD-4, either associated to plasma cell membrane or immobilized on polyvinylidene fluoride (PVDF) strips (Hamon et al., 2004Go), this chemokine has a very weak affinity for soluble PGs (Netelenbos et al., 2003Go). Taken together, our data argue for the specificity of RANTES interaction with the shed ectodomains of SD-1, SD-4, and CD44 solubilized in the HeLa cells culture supernatant.

If the complexes were immobilized on anti-RANTES coated protein-G beads, then treated with heparitinase I and III mixture, SD-1, SD-4 and CD44 binding to RANTES was reversed, since these RANTES-bound PGs were not detected (data not shown). This demonstrates the GAGs-dependence of RANTES binding to the shed PGs.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
SDs bind to various growth factors and cytokines via GAG chains and consequently regulate signal transduction (Bernfield et al., 1992Go, 1999Go; Carey, 1997Go; Couchman et al., 2001Go; Simons and Horowitz, 2001Go; Woods and Couchman, 1998Go; Zimmermann and David, 1999Go). The intact ectodomain of each SD is constituvely shed from cultured cells (Jalkanen et al., 1987Go), as part of normal cell surface HS PG turnover. However, the regulation and the role of this shedding are poorly understood.

The present study was undertaken to investigate whether RANTES directly binds to PGs and to their ectodomains shed from cultured cells and whether this chemokine regulates the shedding process.

Our previous coimmunoprecipitation experiments have suggested that besides its GPCRs, RANTES binds to SD-1 and SD-4 expressed by human monocyte-derived macrophages and HeLa cells expressing or lacking CCR5 (Slimani et al., 2003aGo,bGo). These data indicated, however, that CCR5 is not required for the interaction of RANTES with SD-1 and SD-4. Others have shown that RANTES can be coimmunoprecipitated with CD44, a PG expressed by HeLa cells (Roscic-Mrkic et al., 2003Go); we confirmed these data using primary cells, such as human monocyte-derived macrophages (Slimani et al., 2003aGo).

In accordance with these previous studies, our present data first demonstrate at the molecular level a direct and GAG-dependent-binding of RANTES to electroblotted SD-1, SD-4, and CD44 previously enriched from HeLa cells lysates. We then asked whether RANTES significantly accelerates the respective shedding of SD-1, SD-4, and CD44 from HeLa cells. Ectodomain shedding is frequently a regulated process. However, how these sheddings are regulated has been incompletely investigated. For instance, because SD-1 shedding is involved in diverse pathological events, such as tumor progression, the knowledge of its shedding mechanisms may contribute to the development of diagnostic and therapeutic strategies. Diverse agonists were identified as accelerators of SD-1 and SD-4 shedding (Fitzgerald et al., 2000Go). For instance, shedding accelerated by activation of the epidermal growth factor and thrombin receptor correlates with activation of the ERK/MAPK pathway and does not appear to involve PKC activation (Fitzgerald et al., 2000Go).

In accordance with previous studies (Day et al., 2003Go; Fitzgerald et al., 2000Go; Reiland et al., 1996Go; Subramanian et al., 1997Go), we observe here that SD-1, SD-4, and CD44 are constitutively shed from HeLa cells whether they express CCR5 or not. We then show that RANTES at both physiological and pathological concentrations significantly accelerates SD-1 and SD-4 respective shedding from CCR5-positive HeLa cells. The fact that the same results were observed with RANTES 10–68 or RANTES 1–68 demonstrates that the nine N-terminus amino acids of the chemokine are not involved in these effects. However, the fact that RANTES accelerates the shedding of SD-1 and SD-4 from HeLa cells only if they are CCR5-positive and has no effect on CD44 shedding demonstrates that RANTES stimulation is CCR5-dependent and selective.

The G-protein-coupled pathway activated by RANTES through chemokine receptors involves p44/p42 MAPK activation (Schwabe et al., 2003Go); the compound U0126 is a selective inhibitor of the MAPK family members MEK-1 and MEK-2 (Favata et al., 1998Go), which has little effect on the kinase activities of PKC and others kinases (Favata et al., 1998Go). We report here that as expected, incubation of the CCR5-positive HeLa cells with RANTES induces the phosphorylation of the p44/p42 MAPKs whereas U0126 inhibits this enhanced phosphorylation. The fact that preincubation of the CCR5-positive HeLa cells with both RANTES and U0126 also prevented the accelerated shedding of SD-1 and SD-4 induced by this chemokine strongly suggests the involvement of a CCR5-dependent MAPK pathway in these accelerated sheddings. In addition, the fact that the effect of RANTES on SD-1 and SD-4 shedding from the CCR5-positive HeLa cells was prevented by bisindolylmaleimide I, a inhibitor of PKC, indicates that the action of this chemokine on the acceleration of these PGs shedding also depends from PKC activity. Therefore, the RANTES-induced accelerated shedding of SD-1 and SD-4 is a complex process involving CCR5 and multiple signaling pathways, such as MAPK and PKC.

Moreover, we show here that RANTES induces a decrease in the plasma cell membrane expression of SD-1 and SD-4 by the CCR5-positive HeLa cells, whereas it increases CD44 expression. Therefore, RANTES effects on PGs belonging to the SD family and expressed by CCR5-positive HeLa cells differ from those occurring on others RANTES-interacting PGs such as CD44 expressed by the same cells. These changes in the PGs membrane expression may have functional consequences, on the cell migration, for instance, under physiological or pathological events.

The fact that RANTES did not change the plasma membrane expression of SD-1, SD-4, and CD44 on CCR5-negative HeLa cells also suggests the involvement of CCR5 in these RANTES effects.

PMA does not stimulate the shedding of CD44 from HeLa cells expressing or lacking CCR5, whereas it accelerates the shedding of most PGs, such as SD-1 and SD-4 (Fitzgerald et al., 2000Go; Subramanian et al., 1997Go). Therefore, the regulation mechanisms involved in these PGs shedding differ and the stimulation by PMA of SD-1 or SD-4 shedding is CCR5-independent.

On the other hand, RANTES stimulated the respective increases of SD-1 mRNA and SD-4 mRNA levels, in both HeLa cell types expressing or lacking CCR5. These CCR5-independent stimulations, are therefore followed by RANTES-induced CCR5-dependent molecular events.

Finally, we show that RANTES forms complexes with SD-1, SD-4, and CD44 ectodomains shed from HeLa cells. This was observed if the CCR5-positive HeLa cells were untreated and also if they were stimulated by RANTES. The role of such complexes in the regulation of RANTES effects in physiological or pathological conditions has now to be investigated.

In conclusion, the present data demonstrate that RANTES, acting through a CCR5-dependent pathway, accelerates in a significant manner, the sheddings of SD-1 and SD-4 ectodomains from HeLa cells, and the involvement of both MAPK- and PKC-dependent signaling pathways in these events. They also demonstrate that RANTES forms complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. Whether these complexes lead to down regulation of RANTES signaling can therefore be hypothesized. Moreover, RANTES induces CCR5-dependent decreases in the plasma membrane expressions of SD-1 and SD-4 and on the contrary an increased expression of CD44 on HeLa cells. Additional studies are needed to investigate the consequences of these findings in physiology and diseases.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Cell culture
Transfected CXCR4-positive, CCR5-positive, HeLa cells (HeLa P4) (a gift from A. Benjouad, CERVI, Hôpital Pitié-Salpétrière, Paris) and nontransfected, CXCR4-positive, CCR5-negative, HeLa cells were cultured in Dulbecco minimal essential medium (DMEM; Invitrogen, Paris) containing 10% fetal calf serum (FCS; Biowhittaker, Paris) and 2 mM L-glutamine (Invitrogen) and split twice a week. In addition, the culture medium of the HeLa P4 cells was supplemented by 1 µg/ml puromycin. The HeLa P4 cells are derived from HeLa-clone Z24 (Charneau et al., 1992Go). The P4-CCR5 positive cells were derived from these cells by the transduction of the CCR5 gene using a retroviral vector. It was verified by indirect immunofluorescence assay, followed by both microscopic and cytofluorimetric analysis, that although the nontransfected HeLa cells express CXCR4 as expected (Chang et al., 2002Go), they do not express CCR5. By contrast, the HeLa P4 transfected cells express CXCR4 and CCR5 (data not shown).

Flow cytometry
Adherent HeLa cells were cultured in 24-well flat-bottom plates (at 2 x 105 cells per well) in 1 ml culture medium. After three washes with phosphate buffered saline (PBS) supplemented with 0.05% bovine serum albumin (BSA) (Sigma-Aldrich, Saint Quentin Fallavier, France), cells were incubated for 30 min at 4°C in 150 µl PBS-BSA, supplemented or not with anti-SD-1 antibody DL-101 (mouse IgG-1; clone DL-101; specific for the ectodomain of SD-1 of human origin, Santa Cruz Biotechnology, Santa Cruz, CA), anti-SD-4 antibody 5G9 (clone 5G9; mouse IgG2a; specific for the ectodomain of SD-4 of human origin; Santa Cruz Biotechnology), anti-betaglycan antibodies (goat IgG, R&D systems, Abingdon, UK), anti CD44 antibody (Serotec, Oxford, UK) or their respective isotype-matched control antibodies (mouse IgG1, IgG2a, or goat IgG, Jackson Immunoresearch Laboratories or BD Biosciences Pharmingen, San Jose, CA) (all at 10 µg/ml). After washing, cells were incubated for 30 min at 4°C in 150 µl PBS-BSA supplemented with fluorescein isothiocyanate–labeled goat anti-mouse or mouse anti-goat Ig antibodies (1/20; Pharmingen), fixed in 1% paraformaldehyde (Sigma-Aldrich) in PBS, and scraped.

Cells (10,000) were analyzed by using a FACScan instrument (Becton Dickinson, Le Pont de Claix, France). Results are expressed as the mean fluorescence intensity of the cells. PG expression of the cells, incubated in the presence of RANTES (1–68 or 10–68, devoid of the nine amino acids N-terminus), both synthesized as described (Ylisastigui et al., 1998Go), by C.Vita, CEA, Saclay, France) (at 5–125 nM) or PMA (0.5 µM) (Sigma-Aldrich) was then expressed as the percent change in mean fluorescence intensity (mean ± SEs), as compared with that of unstimulated control cells. The statistical significances of the coupled differences, between data of at least three individuals experiments, were evaluated by the coupled Student t-test.

Preparation of PGs
The PGs from the cells lysates or their shed ectodomains from supernatant culture media were enriched by anion exchange chromatographies. HeLa cells were washed on ice with HEPES saline buffer (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, pH 7.4; all from Sigma-Aldrich) and sonicated thrice for 30 s in lysis buffer (10 mM Tris, 8 M urea, 0.1% [w/v] Triton X-100, 1 mM Na2SO4, 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 8, all from Sigma-Aldrich). In parallel, the cell culture media were collected. Lysates or cell culture media were incubated for 12 h at 4°C with DEAE Sephacel beads (Sigma-Aldrich). The beads were then washed with Tris-buffered saline/EDTA buffer (10 mM Tris, 150 mM NaCl, 0.5 mM EDTA, pH 7.4). Bound PGs were eluted with buffer (100 mM HEPES, 1 M NaCl, 10 mM CaCl2, 20 mM NaOAc, 0.2 mg/ml BSA, 0.6 % [w/v] CHAPS, pH 6.5, all from Sigma-Aldrich). Eluates were diluted with double-distilled H2O to reduce the NaCl concentration to 200 mM. These eluates contained the enriched PGs from the cell lysates or the enriched ectodomains from culture supernatants.

Aliquots of these eluates were then treated with heparitinases I (HS lyase; EC 4.2.2.8; 1 U/ml) and III (heparin lyase; EC 4.2.2.7; 15 U/ml) and chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4; 5 U/ml) (all from Sigma-Aldrich) mixture. Intact and glycosaminidase-treated PGs were electroblotted and characterized as described next.

Binding of biotinylated RANTES to electroblotted PGs
PGs, obtained as just described, were loaded onto 12% sodium dodecyl sylfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels (Invitrogen) under nonreducing conditions and blotted onto PVDF membranes (Amersham Pharmacia Biotech). The membrane was cut in different strips and used for binding assays (Modrowski et al., 2000Go). In some experiments, before the binding assay, electroblotted PGs were digested for 18 h at 37°C with 10 mU/ml heparitinase III (heparin lyase; EC 4.2.2.7), 20 mU/ml heparitinase I (HS lyase; EC 4.2.2.8) and 33 mU/ml chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4) (all from Sigma-Aldrich) diluted in Tris-HCl (20 mM, pH 7.2) supplemented with 1 mM CaCl2, 0.5 mg/ml BSA, and a mixture of protease inhibitors (10 mM PMSF, 25 mM phenanthrolin, 5 mM iodoacetamide; all from Sigma-Aldrich).

Strips were then blocked by incubation for 18 h at 37°C in Tris-buffered saline supplemented (TBS 20 mM) with 5% BSA (w/v) and 0.2% Tween 20 (v/v). Excess BSA was washed out with TBS supplemented with 0.5% BSA and 0.2% Tween 20 (v/v) (Sigma-Aldrich). Strips were incubated for 1 h at room temperature with biotinylated RANTES (RANTES-1-Biotin or RANTES-66-Biotin at 0.05 µg/ml, both synthetized by C. Vita). In some experiments, RANTES 1-Biotin was preincubated, for 2 h at 20°C, with 330 µg/ml of heparin (Sigma-Aldrich). The mix was then incubated with the strips, as just described. It was previously verified that biotin incorporation did not modify the behavior of the chemokine.

After washes with the buffer, strips were reacted with streptavidin-peroxydase (1.5 µg/ml, Sigma-Aldrich) for 60 min at room temperature. After washing, strips were revealed by enhanced chemoluminescence (ECL) detection (Amersham Pharmacia Biotech or Supersignal West Dura Extended, Pierce, Perbio Science, Brebières, France). Alternatively, strips were incubated for 1 h at room temperature with anti-SD-1 DL-101, anti-SD-4 5G9, anti-HS 10E4 or 3G10 monoclonal antibodies (both from Seikagaku, Tokyo) or with their isotypes. After three washes, strips were incubated with horseradish peroxidase (HRP)–labeled anti-mouse IgG (Amersham Pharmacia Biotech)(at 1/5000) and revealed as just described.

Shedding assays
Adherent HeLa cells expressing or lacking CCR5 were cultured in 24-well tissue culture plates. At the time of the assay, culture media were replaced with fresh culture medium supplemented or not by the indicated test agents (0.5 µM PMA; RANTES 1–68 (RANTES); RANTES 10–68, at 0, 5, 50, 125 nM, in the presence or the absence of either a MEK1/2 inhibitor, U0126 at 10 µM; Cell Signaling Technology, Ozyme, France) (Favata et al., 1998Go) or a potent and selective inhibitor of PKC, bisindolylmaleimide I, at 1 µM (Calbiochem) (Toullec et al., 1991Go). Cells were then incubated either for 2 h or 18 h at 37°C in the conditioned media. Aliquots of the cells were labeled with trypan blue exclusion dye and examined by phase microscopy for survival and morphology. Conditioned media were used for dot blot analysis. Cells were analyzed in parallel by flow cytometry after indirect immunofluorescence labeling.

Dot immunoassays
HeLa cells–conditioned media were applied to cationic nylon membrane (Millipore Ny+ or Amersham N+, Amersham Pharmacia Biotech) under mild vacuum in a immunoblot apparatus (slot blot, Amersham Bioscience). This membrane was pretreated by incubation with PBS supplemented with Tris (10 mM). The membrane was then blocked by a 1-h incubation at 37°C in PBS supplemented with 0.5% BSA, 3% nonfat dry milk, and 0.5% Tween 20; washed twice with PBS-BSA 0.5% supplemented with 0.5% Tween 20 (all from Sigma-Aldrich); and then incubated overnight at 4°C, with anti-SD-1 antibody BB4 (mouse IgG-1, specific for the ectodomain of SD-1 of human origin, Serotec, Oxford, UK), anti-SD-4 antibody, anti-CD44 antibody (IgG1; Santa Cruz Biotechnology), or with their respective isotypes (mouse IgG1, IgG2a both from Pharmingen) (all at 0.1 µg/ml). After washing, the membrane was incubated with a 5000-fold dilution of HRP-conjugated anti-mouse IgG (Amersham). Detection was performed using the ECL system. Results were quantified by scanning the exposed X-ray film with an Agfa scanner and using area measurement from Scion imager. Experimental values were within the linear range of the assays. Results are expressed as the amount of PG shed, in relative absorbance units to the value given by the untreated cells (AU). Each point represents the mean±SEs of triplicate determinations of individual experiments. The statistical significances of the coupled differences, between data of at least three individuals experiments, were evaluated by the coupled Student t-test.

Activation of p44/p42 MAPK by RANTES
HeLa P4 cells were washed twice with PBS and maintained in DMEM with 0.1% FCS for 48 h. The cells were then incubated with RANTES (at 125 nM) for 15 min at 37°C. After washing with PBS-orthovanadate (1 mM, Sigma-Aldrich), whole-cell extracts (WCEs) were prepared by lysis of the cells in 20 mM Tris, 150 mM NaCl, 1 mM orthovanadate, NP 40 1%, 10 mM PMSF, 5 mM iodoacetate, 25 mM phenanthrolin, and 20 µg/ml aprotinin. The protein concentration in WCE was determined by the BCA protein assay (Pierce). Equal amounts of total protein extract were separated on 10% SDS–PAGE and transferred to nitrocellulose membrane (Amersham). MAPKs were detected using a 1:1000 dilution of polyclonal antibodies specific for phospho-p44/42 (Thr 202/Tyr 204) or specific for total p44/p42 (all from Cell Signaling Technology). After washing, strips were incubated with HRP-labeled anti-rabbit IgG (Cell Signaling Technology) (at 1/5000) and revealed by ECL reagent.

RNA extraction and amplification by RT-PCR
SD-4 mRNA, SD-1 mRNA, and, to normalize for input of total RNA, glyceraldehyde 3-phosphodehydrogenase (GAPDH) mRNA were quantified by reverse transcription polymerase chain reaction (RT-PCR).

Total cellular RNA was extracted from confluent monolayers of stimulated or unstimulated HeLa cells grown in a six-well tissue culture, using a Qiagen RNA-DNA Mini Kit (Qiagen, France). Reverse transcription was performed using a Advantage RT-for-PCR Kit (BD Biosciences Clontech, France). The following synthetic SD-4 primers were used: upper primerCGA GAG ACT GAG GTC ATC GAC; lower primer: CGC GTA GAA CTC ATT GGT GG. These primers were designed to amplify a 531-bp coding sequence of SD-4. The following SD-1 primers were used: upper primer: TCT GAC AAC TTC TCC GGC TC; lower primer: CCA CTT CTG GCA GGA CTA CA; These primers were designed to amplify a 211-bp coding sequence of SD-1. After 23 cycles of amplification, 30 µl were electrophoresed in 2% agarose and analyzed.

RANTES forms complexes with the shed ectodomains of SD-1, SD-4, and CD44
The enriched ectodomains of PGs, which were shed from the HeLa cells, or the culture supernatants of the HeLa cells (5 ml) were incubated for 18 h at 20°C in the presence of RANTES (at 50–250 nM) and protease inhibitors, followed by incubation for 24 h at 20°C with either anti-RANTES, anti-SD-1 DL-101, anti-SD-4 5G9, or anti-CD44 antibodies–protein-G coated beads, prepared as previously described (Mbemba et al., 1999Go, 2000Go, 2002Go). Alternatively, the cells were cultured for 18 h in the presence of RANTES (at 125 nM). The conditioned supernatant medium was immunoprecipitated as just described. Immunoprecipitated proteins were washed three times with PBS prior to separation by SDS–PAGE (12%). Following electrophoresis, proteins were transferred to PVDF membranes, blocked, and incubated with the appropriate primary antibodies or their respective isotypes as described. The immunoblotted proteins were visualized with HRP-linked secondary antibodies using ECL substrate. When indicated, the enzymatic digestion with heparitinases I and III (1 U/ml and 15 U/ml respectively; both from Sigma-Aldrich), was performed after the immunoprecipitated proteins step, in PBS at 37°C for 2 h. The efficiency of enzymatic digestion was monitored by western blot analysis using anti-HS mAb 10E4.


    Acknowledgements
 
This work was supported by the Agence Nationale de Recherche sur le SIDA, and the Direction de la Recherche et des Enseignements Doctoraux (Ministère de l'Enseignement Supérieur et de la Recherche), Université Paris XIII.


    Footnotes
 
1 These authors contributed equally to this work. Back


    Abbreviations
 
BSA, bovine serum albumin; DMEM, Dulbecco minimal essential medium; ECL, enhanced chemoluminescence; FCS, fetal calf serum; GAG, glycosaminoglycan; GAPDH, glyceraldehyde 3-phosphodehydrogenase; GPCRs, G protein–coupled receptors; HRP, horseradish peroxidase; HS, heparan sulfate; MAPKs, MAP kinases; PBS, phosphate buffered saline; PG, proteoglycan; PKC, protein kinase C; PMA, phorbol 12-myristate13-acetate; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride; RANTES, regulated on activation normal T cell expressed and secreted; RT-PCR, reverse transcription polymerase chain reaction; SD, syndecan; SDS–PAGE, sodium dodecyl sylfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; WCE, whole-cell extract


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 Top
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 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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