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
Received on June 8, 2004; revised on August 3, 2004; accepted on September 3, 2004
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Abstract |
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Key words: chemokine / proteoglycan / RANTES / syndecan-1 / syndecan-4
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Introduction |
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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., 2001; Woods and Couchman, 1998
). 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., 1999
; Clasper et al., 1999
; Zimmermann and David, 1999
). 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., 2003a
,b
), beside CCR5 and CD44 (Roscic-Mrkic et al., 2003
, unpublished data).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., 2003a
, unpublished data).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., 2003a
, unpublished data).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., 1999). It is therefore known that SD-1 and SD-4 are constitutively shed by cultured cells (Jalkanen et al., 1987
) and that this shedding involves release of the soluble ectodomains (Saunders et al., 1989
). Moreover, CD44 shedding by proteolytic processing was also reported (Brennan et al., 1999
; Cichy and Pure, 2004
). 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., 1992
; Jalkanen et al., 1987
). 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., 2000
; Subramanian et al., 1997
). 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.
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Results |
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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., 1996; Hamon et al., 2004
; Kainulainen et al., 1998
; Slimani et al., 2003b
). Both HeLa cell lines used here express CXCR4, SD-2, betaglycan (data not shown) (Chang et al., 2002
; Hamon et al., 2004
), 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., 2003b
). The PGs-enriched fraction obtained from lysates of CCR5-positive or -negative HeLa cells were further characterized (Hamon et al., 2004
) after treatment by heparitinase I, III, and chondroitinase ABC mixture. In such conditions, 32-kDa and 5058-kDa proteins, immunoreactive with both anti-SD-4 5G9 and 3G10 monoclonal antibodies were observed (Figure 1B and data not shown). The 5058-kDa proteins may represent homo- or hetero-oligomers of the SD4-core protein, which is a 32-kDa protein (Oh et al., 1997
). 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., 1992
), 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., 1992
; 1999
).
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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., 1999; Samson et al., 1997
). 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., 2003
). Therefore, we investigated the effects of RANTES (168) or lacking the nine N-terminus amino-acids (1068) 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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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., 1996, 1998
; Schwabe et al., 2003
; Tilton et al., 2000
), and also a protein kinasemediated phosphorylation of four distinct C-terminal serine residues of CCR5 (Pollok-Kopp et al., 2003
).
The compound U0126 inhibits two MAPKs, MEK-1 and MEK-2 (Favata et al., 1998); this inhibition is selective because U0126 has little, if any, effect on the kinase activities of PKC and other kinases (Favata et al., 1998
). 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., 1999
). In the present study, we observe in accordance with others (Schwabe et al., 2003
), 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., 1991), 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., 2000
), 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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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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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.
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Discussion |
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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., 2003a,b
). 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., 2003
); we confirmed these data using primary cells, such as human monocyte-derived macrophages (Slimani et al., 2003a
).
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., 2000). 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., 2000
).
In accordance with previous studies (Day et al., 2003; Fitzgerald et al., 2000
; Reiland et al., 1996
; Subramanian et al., 1997
), 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 1068 or RANTES 168 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., 2003); the compound U0126 is a selective inhibitor of the MAPK family members MEK-1 and MEK-2 (Favata et al., 1998
), which has little effect on the kinase activities of PKC and others kinases (Favata et al., 1998
). 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., 2000; Subramanian et al., 1997
). 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.
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Materials and methods |
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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 isothiocyanatelabeled 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 (168 or 1068, devoid of the nine amino acids N-terminus), both synthesized as described (Ylisastigui et al., 1998), by C.Vita, CEA, Saclay, France) (at 5125 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 sylfatepolyacrylamide gel electrophoresis (SDSPAGE) 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., 2000). 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 168 (RANTES); RANTES 1068, 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., 1998) or a potent and selective inhibitor of PKC, bisindolylmaleimide I, at 1 µM (Calbiochem) (Toullec et al., 1991
). 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 cellsconditioned 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% SDSPAGE 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 50250 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 antibodiesprotein-G coated beads, prepared as previously described (Mbemba et al., 1999, 2000
, 2002
). 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 SDSPAGE (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.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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