Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12 ´ Nathalie Charnaux1,2*, Severine Brule1,2*, Morgan Hamon1, Thomas Chaigneau1, Line Saffar1, Catherine Prost1, Nicole Lievre1 and Liliane Gattegno1,2 ´ ´ ´ ´ Laboratoire de Biologie Cellulaire, Biotherapies Benefices et Risques, UPRES 3410 Universite Paris XIII, Bobigny, France ˆ Hopital Jean Verdier, Bondy, France Keywords CXCR4; proteoglycan; SDF-1 ⁄ CXCL12; syndecan-4 Correspondence L Gattegno, Laboratoire de Biologie ´ ´ ´ Cellulaire, Biotherapies Benefices et ´ Risques, UPRES 3410 Universite Paris XIII, 74, rue Marcel Cachin, 93017, Bobigny, ˆ France, Hopital Jean Verdier, 93017, Bondy, France Fax: +33 48026503 Tel: +33 48387752 E-mail: liliane.gattegno@jvr.ap-hop-paris.fr *These authors contributed equally to this work (Received 18 January 2005, accepted 21 February 2005) doi:10.1111/j.1742-4658.2005.04624.x Stromal cell-derived factor-1 (SDF-1) ⁄ CXCL12, the ligand for CXCR4, induces signal transduction We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan We also demonstrated the occurrence of a CXCL12independent heteromeric complex between CXCR4 and syndecan-4 However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4 Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4 Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44 ⁄ p42 mitogen-activated protein kinase and Jun N-terminal ⁄ stress-activated protein kinase activation by action of CXCL12 on HeLa cells However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events Chemokines are low molecular mass proteins mediating several functions such as hematopoiesis regulation, leukocyte maturation, angiogenesis, T and B lymphocytes trafficking, homing and lymphoid tissues development [1–3] Stromal cell-derived factor-1 (SDF-1) ⁄ recently renamed CXCL12 [4], is the only known ligand for CXCR4 [5,6] SDF-1 and CXCR4 are constitutively expressed in various tissues [7] and are implicated in several diseases CXCR4 is involved in HIV infection and pathogenesis [5,8] SDF-1 and CXCR4 also regulate Abbreviations dsRNA, double-stranded RNA; FBS, fetal bovine serum; GAG, glycosaminoglycan; HS, heparan sulfate; JNK ⁄ SAPK, Jun N-terminal ⁄ stressactivated protein kinase; MAPK, mitogen-activated-protein kinase; PFA, paraformaldehyde; PMA, phorbol 12-myristate-13-acetate; PG, proteoglycan; Ptyr, tyrosine phosphorylated; SDF, stromal cell-derived factor; SD, syndecan FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS 1937 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 embryonic development [9] Much of the heparan sulfate (HS) at the cell surface is derived from the syndecan (SD) family of transmembrane proteoglycan (PG) [10] The SDs bind a variety of growth factors, cytokines, proteases, antiproteases and cell adhesion molecules [10,11]; they are individually expressed in distinct cell-, tissue-, and development-specific patterns [12], and show cell-specific variations in the structure of their HS chains [13] SDs may regulate ligand-dependent activation of cell surface growth factor receptors by several potential mechanisms [10,11,14] SD-4 is one of the principal HS carrying protein on cell surfaces [15,16] We recently showed that SDF-1 binds to high- and low-affinity sites on HeLa cells and forms complexes on these cells and on human primary lymphocytes and macrophages, which comprise CXCR4, as expected, and also SD-4 [17], but not SD-1, SD-2, betaglycan or CD44 ([17] and unpublished data) Moreover, we recently demonstrated the occurrence of an SDF-1independent heteromeric complex on the plasma membrane of these cells, which comprises CXCR4 and SD-4 but not SD-1, SD-2, CD44 or beta-glycan [17] This suggested that SDF-1 may bind both the PG SD-4 and its G-protein-coupled receptor (GPCR), CXCR4 However, our previous data have shown that while glycosaminidases pretreatment of primary macrophages decreases the binding of SDF-1 to CXCR4, such treatment had no effect on the chemokine binding to CXCR4 expressed by the HeLa cell line [17] This has suggested that while SD-4 may serve as a binding anchor for SDF-1 on primary macrophages to enable the chemokine to interact with CXCR4, this was not true if HeLa cells were tested The present study was designed to test whether SD-4 functions as a specific SDF-1 signaling molecule Therefore, we first determined whether SDF-1 directly binds SD-4 and the glycosaminoglycan (GAG)dependency of this binding Because protein phosphorylation plays a critical role in the generation of intracellular signals in response to external stimuli, we then investigated whether SD-4 becomes tyrosine phosphorylated (Ptyr) upon SDF-1 stimulation of HeLa cells, and whether, in these conditions, tyrosinephosphorylated SD-4 is physically coassociated with tyrosine-phosphorylated CXCR4, and what the GAGdependency of these events is Finally, we asked whether SD-4 is involved in other biochemical signals induced by SDF-1 By specifically reducing SD-4 expression using RNA interference, or by reducing the HS expressed at the plasma membranes of HeLa cells by the use of heparitinases I and III, we analyzed the respective roles of SD-4 and HS in transduction pathways induced by SDF-1 on these cells 1938 N Charnaux et al Results SDF-1 directly binds to SD-4 The HeLa cells used in the present study express CXCR4, SD-2, beta-glycan (data not shown) [17,18], CD44, SD-1 and SD-4 (Fig 1A), as assessed by flow cytometry analysis after indirect immunofluorescence A a b c B Fig PGs on HeLa cells (A) Cell surface expression of SD-1, SD-4 and CD44 on HeLa cells HeLa cells (5 · 105) were stained for FACS analysis with anti-(SD-1) DL-101 mAb (a), anti-(SD-4) 5G9 mAb (b) or anti-CD44 mAb (c) (thick lines) Reactivity was compared to an isotype-matched control monoclonal antibody (a,b,c, dotted lines) (B) Immunoblot analysis of PGs from HeLa cells HeLa cells were lysed in the presence of Triton X-100 and urea PGs (from · 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 mAb (lane 1) or the isotype, IgG2b (lane 2) The respective immunoreactivity with anti-(SD-1) DL-101, anti(SD-4) 5G9, anti-CD44 mAbs, or anti-(SD-2) Igs are represented by arrows Data are representative of three individual experiments FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS N Charnaux et al labeling The core proteins of most PGs enriched from HeLa cells lysates were analyzed [17] after heparitinase I and III and chondroitinase ABC treatment to detect their apparent molecular masses Proteins of 32 kDa and 50–58 kDa, immunoreactive with anti-SD-4 5G9 and 3G10 mAbs, were observed (Fig 1B) The 50– 58 kDa proteins may represent, in accordance with other studies, homo- or hetero-oligomers of the SD-4 core protein, which is a 32 kDa protein [19] Other PGs were also detected: 34 kDa proteins immunoreactive with both anti-SD-2 mAbs and mAb 3G10, 45- and 90 kDa proteins immunoreactive with antiSD-1DL-101 and 3G10 mAbs (the 90 kDa ones probably being dimers of the 45 kDa ones), and 60 kDa proteins immunoreactive with anti-CD44 and 3G10 mAbs (Fig 1B) All these apparent molecular masses are close to the predicted ones [9] These PGs were glycanated, as mAb 3G10 reacts with an epitope including a terminal unsaturated uronic acid residue, which is unmasked after HS removal by heparitinases treatment [20] Native PGs may migrate in a diffuse high molecular mass distribution on SDS ⁄ PAGE Using the respective specific Abs, glycanated PGs migrate as follows: SD-4 as a 100–250 kDa broad smear, SD-1 as a single 98 kDa band, CD44 as a 110 kDa band, SD-2 as a 50 kDa protein Beta-glycan migrates as two broad bands of 55 and 100 kDa, respectively (Fig 2, lanes 1–5) No immunoreactivity was detected with the isotypes (data not shown) The fact that all these PGs were also immunoreactive with anti-HS mAb 10E4, Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 but not with its isotype, demonstrates their glycanation (Fig 2, lane and data not shown) Biotinylated SDF-1a bound to the broad smear of 100–250 kDa, characterized as glycanated SD-4, but did not bind to SD-1, SD-2, beta-glycan or CD44 (Fig 2, lane vs 1–5) Heparitinase I and III, and chondroitinase ABC pretreatment of the strips abolished the binding of electroblotted PGs to anti-HS mAb 10E4 (Fig 2, lane vs 6), and strongly decreased that of biotinylated SDF-1a to SD-4 (Fig 2, lane vs 7), but did not change SD-4 binding to anti-SD-4 mAb 5G9 (specific for the core protein of SD-4)(data not shown) This demonstrated that (a) the heparitinases treatment was efficient; (b) SD-4 was still present on the polyvinylidene difluoride membrane (data not shown); and (c) the direct binding of SDF-1 to SD-4 was GAG dependent Confocal microscopy analysis showed that fluorescently labeled biotinylated SDF-1a colocalizes with SD-4 on the plasma membranes of these cells, as assessed by the yellow (red-green colocalization) staining (Fig 3A, and data not shown) This association was further analyzed by electron microscopy (Fig 3B) Beads at the cell surface were counted and considered as associated when the distance between them was less than 15 nm Forty per cent of the beads that labeled SD-4 were associated with 45% of the beads that labeled SDF-1a, while no association of SDF-1a with SD-1 was detected Controls, run without biotinylated SDF-1a or with the isotypes, were not stained (data not shown) Fig SDF-1 binds to SD-4 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-4 5G9 mAb (lane 1), anti-(SD-1) DL-101 mAb (lane 2), anti-CD44 mAb (lane 3), anti-(SD-2) Igs (lane 4), anti-(beta-glycan) Igs (lane 5), anti-HS 10E4 mAb (lane 6), biotinylated SDF-1a (lane 7) Alternatively, strips were treated with heparitinases I, III mixture and revealed with anti-HS mAb 10E4 (lane 8) or biotinylated SDF-1a (lane 9) Data are representative of three individual experiments FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS 1939 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 A Syndecan-4 SDF-1α N Charnaux et al Merged B SDF-1 induces the tyrosine phosphorylation of CXCR4 and the homo- or hetero-oligomerization of this GPCR on HeLa cells SDF-1-activated or nonactivated HeLa cell lysates were either immunoprecipitated with anti-CXCR4 mAb G19 then blots were developed with anti-Ptyr mAb 4G10, or immunoprecipitated with anti-Ptyr mAb 4G10 then blots were developed with anti-CXCR4 mAb 12G5 A protein was observed at 48 kDa, and several others of apparent molecular masses > 48 kDa All amounts of these tyrosine-phosphorylated proteins were significantly increased upon SDF-1 stimulation of the cells (Fig 4A, lane vs 1, and lane vs 3) These increases were not significant if the cells were stimulated with nm SDF-1, and strongly significant for a cell stimulation with 125 nm SDF-1 These increases were marginally observed if the cells were stimulated for or 30 in the presence of 125 nm SDF-1 and were strongly significant after 10 of incubation of the cells with 125 nm of the chemokine (Fig 4A and data not shown) Among these tyrosine phosphorylated proteins, those immunoreactive with anti-CXCR4 mAb 12G5 probably represent, respectively, CXCR4 monomers and homo- or hetero-oligomers (Fig 4A, lane 4) Residual phosphorylation of CXCR4 in unstimulated cells was 1940 Fig SDF-1 colocalizes with SD-4 on HeLa cells (A) HeLa cells were double stained with fluorescently labeled biotinylated SDF-1a (green) and anti-(SD-4) mAb 5G9 (red) Confocal microscopy analysis shows the colocalization of biotinylated SDF-1a with SD-4, as assessed by the yellow (red-green) colocalization, suggesting the clustering of SDF-1 and SD-4 Data are representative of three individual experiments Bar ¼ lm (B) HeLa cells were double-stained with biotinylated SDF-1a and with anti-(SD-4) mAb Stainings were revealed with streptavidin-15 nm colloidal gold particles or anti-mouse Ig bound to nm colloidal gold particles, respectively Black arrows show colocalization of 6- and 15-nm colloidal gold particles Bar ¼ 100 nm (initial magnification · 27 500) Data are representative of three individual experiments detected (Fig 4A, lanes and 3), as reported previously [21–23] SDF-1 also induces the tyrosine phosphorylation of SD-4 on HeLa cells and tyrosine phosphorylated SD-4 is physically associated to tyrosine phosphorylated CXCR4 The anti-CXCR4 G19 IP of SDF-1-stimulated cell lysates revealed with anti-Ptyr mAb 4G10, just described, was also characterized by a 110–200 kDa broad smear, which was marginally revealed if the cells were not stimulated (Fig 4A, lane vs 1) and was not detected if the anti-Ptyr 4G10 IP was revealed with anti-CXCR4 mAb 12G5 (Fig 4A, lane vs 2) This suggests that it represents proteins which are physically associated to CXCR4 and are tyrosine phosphorylated when the cells are stimulated by SDF-1 To characterize these proteins, the SDF-1-unactivatedand SDF-1-activated HeLa cell lysates were immunoprecipitated in parallel with anti-Ptyr mAb 4G10 and blots were developed with several different anti-PG Abs: anti-SD-4 mAb 5G9, anti-SD-1 mAb DL-101, anti-SD2 Abs or anti-beta-glycan Abs (Fig 4B and data not shown) The tyrosine phosphorylated smear described above was only significantly observed when the antiPtyr 4G10 IP from SDF-1-activated HeLa cell lysates FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS N Charnaux et al A Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 B C D Fig SDF-1 induces the tyrosine-phosphorylation of SD-4 on HeLa cells Confluent serum-starved HeLa cells were either stimulated (+) or not (–) with SDF-1a Equal amounts of proteins from whole cell extracts were immunoprecipitated with the indicated antibodies and equivalent amounts of IP samples were separated on 12% SDS ⁄ PAGE and immunoblotted using the indicated mAb or polyclonal antibodies (A) HeLa cells were stimulated (+) (lanes 2,4) or not (–) (lanes 1,3) for 10 with 125 nM SDF-1a Cell lysates were immunoprecipitated either with anti-CXCR4 Igs G19 (lanes 1,2) or anti-Ptyr mAb 4G10 (lanes 3,4) Western blots were developed, respectively, with anti-Ptyr mAb 4G10 (lanes 1,2) or anti-CXCR4 mAb 12G5 (lanes 3,4) (B) HeLa cells were stimulated (+) (lanes 2,3,4,6,8) or not (–) (lanes 1,5,7) with 125 nM SDF-1a for the indicated time Cell lysates were immunoprecipitated with anti-Ptyr mAb 4G10 (lanes 1–8) Western blots were developed with anti-(SD-4) mAb 5G9 (lanes 1–4), anti-b-glycan Abs (lanes 5,6) or anti-(SD-2) Igs (lanes 7,8) (C,D) The intensities of the phosphorylated bands shown in A and B (lanes 1–4) were quantified in absorbance units by densitometric scanning and analyzed with SCION IMAGER They were expressed as ratios of the data observed for the SDF-1 stimulated cells relative to the untreated control cells Each bar represents the mean ± SE of triplicate determinations of an individual experiment The significance of the differences as compared with untreated control cells was assessed using Student’s t-test: **P < 0.05 The position of immunoglobulin chains is indicated by a star Protein bands with changes in tyrosine phosphorylation state are indicated by arrows was revealed with anti-SD-4 mAb 5G9 (Fig 4B, lane vs 6, and data not shown); it was marginally observed if the cells were not stimulated (Fig 4B lane 1) This increase of the tyrosine-phosphorylation of SD-4 induced by SDF-1 on HeLa cells is time and concentration-dependent: it was marginal if the cells were incubated for or 30 with 3, 50 or 125 nm of SDF-1, and significant if the cells were incubated for 10 with 125 nm SDF-1 (Fig 4B, lanes vs 2, and data not shown) These latter conditions were therefore used for the following IPs To further demonstrate the occurrence of tyrosine-phosphorylated SD-4, the SDF-1unactivated- and SDF-1-activated HeLa cell lysates were precipitated with anti-SD-4 mAb 5G9 and developed with anti-Ptyr mAb 4G10 (Fig 5A, lanes and 2) To confirm equal loading of the samples, the 5G9 IPs FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS were stripped and reprobed with anti-SD-4 mAb 5G9 (Fig 5A, lanes and 6) The phosphorylated 110– 200 kDa smear was revealed with anti-Ptyr mAb 4G10 in the electroblotted IP of the SDF-1-stimulated cell lysates (Fig 5A, lane 2) This smear was marginally revealed in the unstimulated cells (Fig 5A, lane 1) These data strongly indicate that SDF-1 induces a rapid and significant increase in the tyrosine phosphorylation of SD-4 on HeLa cells and that a physical association of tyrosine phosphorylated CXCR4 with tyrosine phosphorylated SD-4 occurs The protein core of tyrosine phosphorylated SD-4 was examined in parallel after digestion of the GAGs chains (Fig 5B) For this purpose, the anti-Ptyr 4G10 IPs and the anti-SD-4 5G9 IPs of the SDF-1-unstimulated and stimulated HeLa cells were treated with a 1941 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 N Charnaux et al A C B Fig Heparan sulfate is involved in the tyrosine phosphorylation of SD-4 induced by SDF-1 on HeLa cells (A) Upper panel: HeLa cells were either stimulated (+) (lanes and 4) or not (–) (lanes and 3) for 10 with 125 nM SDF-1a In some experiments, cells were pretreated in parallel with heparitinases I and III mixture (lanes and 4) Lysates were then immunoprecipitated with anti-(SD-4) mAb 5G9 Western blots were developed with anti-Ptyr mAb 4G10 (lanes 1–4) Lanes and confirm the equal loading of samples by reprobing the polyvinylidene difluoride membrane with anti-SD-4 5G9 mAb The position of the immunoglobulin chains is indicated by a star (B) HeLa cells were stimulated (+) (lanes 2, 4, and 8) or not (–) (lanes 1, 3, and 7) for 10 with 125 nM SDF-1a Cells lysates were immunoprecipitated with antiPtyr 4G10 mAb (lanes 1–4) or with anti-SD-4 5G9 mAbs (lanes 5–8) The IPs were treated with heparitinases I, III, and chondroitinase ABC Western blots were developed, respectively, with anti-SD-4 5G9 mAb (lanes and 2), anti-(SD-1) DL-101 mAb (lanes and 4), anti-Ptyr 4G10 mAb (lanes and 6) or the isotype IgG2b (lanes and 8) (C) Upper panel: HeLa cells were stimulated (+) (lanes and 3) or not (–) (lane 1) for 10 with 125 nM SDF-1a In some experiments, cells were pretreated, in parallel, with heparitinases I and III (lane 3) Lysates were then immunoprecipitated with anti-(SD-4) mAb 5G9 Western blots were developed with anti-CXCR4 mAb 12G5 Lower panels in (A) and (C): The data shown in (A) (lanes 1–4) and in (C) were quantified in absorbance units by densitometric scanning and analyzed with SCION IMAGER They were expressed as the ratios of the data observed for the SDF-1 stimulated cells relative to those observed for the corresponding unstimulated, control cells Each bar represents the mean ± SE of triplicate determinations of an individual experiment The significance of the differences as compared either with controls or with heparitinase-treated cells was assessed using a t-test **P < 0.05 1942 FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS N Charnaux et al mixture of heparitinases I and III, and chondroitinase ABC, and then eluted from the beads The eluates were electroblotted and revealed, respectively, with anti-SD-4 mAb 5G9 and anti-Ptyr mAb 4G10 Proteins of 50–55 kDa which increased significantly after stimulation of the cells by SDF-1 were revealed No immunoreactivity was detected using either the isotype or anti-SD-1 mAb DL-101, anti-SD-2 and anti-(betaglycan) Igs (Fig 5B and data not shown) We then used coimmunoprecipitation experiments to further analyse the physical association of CXCR4 and SD-4 The anti-SD-4 5G9 IPs of unstimulated as well as SDF-1-stimulated HeLa cells lysates, respectively, were characterized by the presence of 48 kDa proteins and of several other minor proteins of apparent molecular masses > 48 kDa, all immunoreactive with 12G5 (Fig 5C, lanes and 2) Therefore, the SDF-1independent heteromeric complex between CXCR4 and SD-4 (Fig 5C, lane 1) is still present if the cells are stimulated by the chemokine (Fig 5C, lane vs 1) The tyrosine phosphorylation of SD-4 induced by SDF-1 on HeLa cells depends on the HS chains of this PG To examine whether the tyrosine phosphorylation of SD-4 induced by SDF-1 on HeLa cells depends on HS, we treated these cells with mixtures of heparitinase I and III prior to their stimulation by SDF-1 To preserve cell viability, concentrations of heparitinases were lower than those used to treat the IPs The efficiency of the enzymes was investigated: if the cells were incubated in enzyme-free medium and then stimulated with SDF-1, the 5G9 IPs revealed with 10E4 showed, as expected, the 110–200 kDa broad smear, described above; however, if the cells were pretreated with heparitinases I and III, this smear was no longer present (data not shown) Moreover, this heparitinases pretreatment of the cells prevented in a significant manner the tyrosine-phosphorylation of SD-4 induced by SDF-1, as assessed by the anti-SD-4 5G9 IPs revealed with anti-Ptyr 4G10 mAb (Fig 5A, lane vs 3) In these experiments, the apparent relative molecular masses of most tyrosine-phosphorylated SD-4 molecules were also decreased, as expected [17] (Fig 5A, lanes and vs 2) The homo- or hetero-oligomerization of CXCR4 induced by SDF-1 on HeLa cells is prevented by heparitinases I and III pretreatment of these cells Heparitinases I and III pretreatment of the HeLa cells also significantly prevented the homo- or hetero- oligoFEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 merization of CXCR4 induced by SDF-1 on HeLa cells, as assessed by the anti-SD-4 5G9 IP of the cell lysates revealed with anti-CXCR4 mAb 12G5 (Fig 5C, lane vs 2) This indicates that the HS-dependent binding of SDF-1 to SD-4 enables the chemokine to induce the homo- or hetero-oligomerization of its GPCR The physical association of tyrosine-phosphorylated SD-4 with tyrosine phosphorylated CXCR4 does not depend on GAGs chains When anti-CXCR4 G19 IPs of the SDF-1 stimulated cell lysates were treated with heparitinases I and III, and chondroitinase ABC mixture, both SD-4 and CXCR4 remained on the beads, as assessed by their respective revelation with 12G5 and 5G9 (data not shown) This suggests that GAG-dependent interactions are not involved in these physical associations Finally, in all the experiments described above, results of immunoprecipitation of cell lysates with isotype-matched control antibodies (data not shown) rule out nonspecific protein association with membrane components under our experimental conditions The activation of p44/p42 MAPK and JNK/SAP kinase by SDF-1 on HeLa cells is HS- and SD-4- dependent To analyze some of the transduction pathways induced by SDF-1 on HeLa cells, whole cell extracts from either unstimulated or stimulated HeLa cells were electroblotted and revealed using phospho-specific antip44 ⁄ p42 mitogen-activated protein kinase (MAPK) or anti-p46 ⁄ p54-Jun N-terminal ⁄ stress-activated protein kinase (JNK ⁄ SAP kinase) Abs, respectively Parallel immunoblottings with anti-total polyclonal Abs confirmed equal loading of the samples (Fig 6) As expected [24–26], SDF-1a and phorbol 12-myristate13-acetate (PMA) induced a rapid activation of p44 ⁄ 42 MAPK and JNK ⁄ SAP kinase signaling in HeLa cells by increasing phosphorylations of the respective proteins (Fig 6) This effect was time and concentrationdependent: It rose from nm up to 125 nm SDF-1a and if the time of incubation with the chemokine was enhanced from to 15 On the contrary, these phosphorylations decreased if the time of incubation with the chemokine was enhanced up to 30 (Fig 6A) According to these results, the cells were incubated for 15 in the presence of 125 nm of SDF-1 in the following experiments (Fig 6B) In these conditions, pretreating these cells with heparitinases I and III significantly decreased these SDF-1-induced phosphorylations (Fig 6B) (P < 0.05) 1943 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 N Charnaux et al A B 300 200 ** 100 phosphorylation level (% versus control) phosphorylation level (% versus control) 400 300 200 ** 100 Fig Heparan sulfate is involved in the activation of MAPK induced by SDF-1 stimulation of HeLa cells (A) Serum-starved HeLa cells were either stimulated or not with nM or 125 nM SDF-1a for 5, 15 and 30 min, and then analyzed for p44 ⁄ p42 MAPK and JUN ⁄ SAPK activations (B) Upper panel: Untreated (–) or heparitinases I- and III-treated (+) HeLa cells were either stimulated or not for 10 with PMA (0.5 lM) or SDF-1a (125 nM).Whole cell extracts were separated on 12% SDS ⁄ PAGE and immunoblotted using either phosphospecific anti-(p44 ⁄ p42 MAPK) or phosphospecific p46 ⁄ p54-SAPK ⁄ JNK rabbit polyclonal antibodies Parallel immunoblottings with anti-(total p44 ⁄ p42 MAPK) or anti(total p46 ⁄ p54-SAPK ⁄ JNK) polyclonal antibodies, respectively, confirmed equal loading of samples Lower panel in (B): The results were quantified by densitometric scanning and analyzed with SCION IMAGER For each lane, data were expressed as p44 ⁄ p42 or SAPK ⁄ JNK phosphorylated proteins over total proteins in absorbance units The amount of MAPK (p44 ⁄ p42 or SAPK ⁄ JNK) phosphorylation in the SDF-1-stimulated cells was calculated according to the level of phosphorylated MAPK proteins in unstimulated control cells, which was considered as 100% Each bar represents the mean ± SE of triplicate determination of an individual experiment The significance of the differences between the SDF-1-stimulated cells and the corresponding heparitinases treated cells was assessed using a t-test **P < 0.05 As expected [21,26,27], SDF-1 also stimulates intracellular calcium mobilization in HeLa cells (Fig 7A) However, enzymatic removal of HS from the surface of these cells did not affect this increased fluorescence intensity observed in dye-loaded cells (mean ± SE ¼ 101 ± 21, n ¼ 30) as compared to untreated control cells (mean ± SE ¼ 97 ± 16, n ¼ 32), or the percentage of SDF-1 responding cells (Fig 7A,B and data not shown) 1944 Transfection of HeLa cells with SD-4 double-stranded RNA (SD-4 dsRNA) resulted, as expected, in a SD-4 mRNA downregulation reaching 80% reduction on day 3, while the mRNAs of SD-1, SD-2 and CXCR4 were not changed (Fig 8A) Moreover, when measuring the expressions of these proteins by FACS in these transfected cells, we found a 65% downregulation of SD-4 expression, while SD-1, SD-2, beta-glycan or CXCR4 expressions remained unchanged as expected FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS N Charnaux et al Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 A B heparitinases I and III treated HeLa cell untreated HeLa cell 120 fluorescence intensity fluorescence intensity 120 60 0 50 60 100 time (sec) 120 mock-transfected HeLa cell 60 50 100 time (sec) (Fig 8B–D and data not shown) To monitor the sequence specificity for SD-4 RNA interference, mutSD-4 dsRNAs was used as a control The mutSD4 dsRNA construct caused no significant reduction of SD-4 mRNA and protein expressions, concordant with previous reports on RNA interference methodology [28,29] (Fig 8A and data not shown) (P ¼ 0.11) We then observed that both p44 ⁄ p42 MAPK and JNK ⁄ SAP kinase activations were significantly reduced after the knockdown of SD-4 upon SDF-1a stimulation, as compared with the data observed in mocktransfected cells and in cells transfected with mutSD-4 dsRNA, respectively (Fig 8E) (P < 0.05) By contrast, under the same conditions, no change in the Ca2+ mobilization induced by SDF-1 after the knockdown of SD-4 on HeLa cells was observed (Fig 7C,D) Discussion CXCR4 and SDF-1 play pivotal roles in many diseases [5–8,30–32] SDF-1 binding to GAGs, especially HS, has been demonstrated [17,33,34] Moreover, SDF-1 forms complexes on CXCR4-positive cells with CXCR4 as expected and also with SD-4, but not with SD-1, SD-2, beta-glycan or CD44 [17] Furthermore, an SDF-1-independent heteromeric complex between CXCR4 and SD-4 occurs on these cells, but not with SD-1, SD-2, beta-glycan or CD44 [17] Therefore, FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS D 100 SD-4 dsRNA HeLa cell 120 fluorescence intensity C fluorescence intensity Fig Heparan sulfate is not involved in intracellular Ca2+ mobilization induced by SDF1 on HeLa cells Untreated HeLa cells (A), heparitinases I- and III-treated HeLa cells (B), mock-transfected HeLa cells (C), or SD-4 dsRNA transfected HeLa cells (D) were loaded for 30 with Fluo-3 and then stimulated with SDF-1a (125 nM), as indicated by black arrows The plots show the variations of the fluorescence intensity (expressed in arbitrary units), measured overtime within the analyzed cells Data are representative of three individual experiments 50 time (sec) 60 0 50 100 time (sec) SDF-1 may bind both its GPCR CXCR4 and SD-4 However, whether SDF-1 directly binds SD-4 has not been demonstrated previously We show here a direct binding of SDF-1 to electroblotted SD-4 enriched from HeLa cell lysates The fact that no binding of the chemokine to SD-1, SD-2, beta-glycan or CD44 was detected strongly argues for the selectivity of this binding We then examined whether SDF-1 is associated with SD-4 at the plasma membranes of intact HeLa cells By using both confocal and electron microscopy analysis, we show strong evidence for the occurrence of a colocalization between SDF-1 and SD-4 at the HeLa cell plasma membrane The fact that in the same conditions, no colocalization of SDF-1 with another PG, SD-1, was observed, argues further for the selectivity of this association Therefore, our findings observed at the molecular level were strengthened by experiments performed at the cellular level Thereafter, we asked whether GAGs are involved in SDF-1 binding to SD-4 By pretreating the electroblotted PGs from the HeLa cells with heparitinase I and III and chondroitinase ABC mixture, we demonstrate the strong GAG dependency of this binding However, our data not rule out the additional involvement of protein–protein interactions between SDF-1 and the SD-4 core protein Indeed, while the SD core proteins share a high degree of conservation in their short cytoplasmic and transmembrane domains, in contrast their 1945 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 A B N Charnaux et al C 32 32 IgGl (SD-4ds RNA) SD-1 (SD-4 ds RNA) IgGl (mocktransfected) 0 10 101 102 103 100 SD-1 (mocktransfected) 101 102 103 104 E D 32 0 10 101 102 103 104 Fig SD-4 is involved in SDF-1 activation of MAPK pathways HeLa cells were transfected with either SD-4 dsRNAs or MutSD-4 dsRNA or were mock-transfected (A) Left panel: HeLa cells were analyzed for SD-4, SD-1, SD-2, CXCR4 specific mRNA, by semiquantitative RT-PCR, days post transfection To normalize for input of total RNA, GAPDH mRNA was also determined Right panel: SD-4 mRNA levels were quantified by densitometric scanning and analyzed with SCION IMAGER Results are depicted relative to mock-transfected control Each bar represents the mean ± SE of triplicate determination of an individual experiment The significance of the differences as compared to mocktransfected control cells was assessed using a t-test **P < 0.05 (B, C, D) HeLa cells were analyzed for (B) SD-4 (C) SD-1 and (D) CXCR4 protein expressions by FACS analysis, days post transfection Reactivity was compared to an isotype-matched control mAb (E) Upper panel: HeLa cells were treated for 15 with 125 nM SDF-1a, days post-transfection Whole cell extracts were separated on 12% SDS ⁄ PAGE and analyzed by immunoblot using phosphospecific anti-(p44 ⁄ p42 MAPK) or phosphospecific p46 ⁄ p54-SAPK ⁄ JNK polyclonal rabbit antibodies, respectively Parallel immunoblotting with anti-(total p44 ⁄ p42 MAPK) or anti-(total p46 ⁄ p54-SAPK ⁄ JNK) polyclonal antibodies was performed to confirm equal loading of samples Lower panel: The results were quantified by densitometric scanning and analyzed with SCION IMAGER For each lane, data were expressed as p44 ⁄ p42 MAPK or SAPK ⁄ JNK phosphorylated proteins over total proteins in absorbance units The amount of MAPK (p44 ⁄ p42 or SAPK ⁄ JNK) phosphorylations in the SDF-1-stimulated cells was calculated according to the level of phosphorylated MAPK proteins in untreated control cells, which was considered as 100% Each bar represents the mean ± SE of triplicate determination of an individual experiment The significance of the differences between the phosphorylation states of the SDF-1a-stimulated, SD-4dsRNA- transfected cells and those of the SDF-1a-stimulated, mock-transfected cells was assessed using a t-test **P < 0.05 extracellular domains are divergent with the exception of consensus sites for GAG attachment [15,35] The participation of the SD-4 ectoplasmic domain in SDF-1 binding raises the question whether this binding is accompanied by intracellular modifications of SD-4 such as tyrosine phosphorylation, which plays critical role in a variety of cellular processes We have therefore asked whether SD-4 functions as an SDF-1 signaling molecule For this purpose, we investigated whether SDF-1 stimulation of HeLa cells induces an increase in the tyrosine phosphorylation of SD-4, besides that of CXCR4 which has already been reported [21–23] The SD cytoplasmic domains contain four conserved tyrosine residues, two of which are in favorable sequences 1946 for phosphorylation [36] Endogenous tyrosine phosphorylation of SDs has already been detected while most cell surface SDs are phosphorylated following treatment with the tyrosine phosphatase inhibitor pervanadate [37] Tyrosine phosphorylation of the SD cytoplasmic domain may be a common mechanism for regulating SD activity In this study, immunoprecipitation experiments using anti-Ptyr, anti-CXCR4 and anti(SD-4) mAbs show for the first time that besides the tyrosine phosphorylation of CXCR4, tyrosine phosphorylation of SD-4 occurs in response to SDF-1 stimulation of HeLa cells This tyrosine phosphorylation depends on the time of incubation of the cells with the chemokine: marginal for 2-min incubation, significant FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS N Charnaux et al for 10-min incubation in the presence of 125 nm SDF1 It also depends on the concentration of the chemokine and is highly significant when 125 nm SDF-1 is used, and marginal for and 50 nm concentrations of the chemokine As SDF-1 stimulation of HeLa cells did not induce the increase in the tyrosine phosphorylation of other PGs, such as SD-1, SD-2 or beta-glycan, this indicates the selectivity of this process However, in agreement with other results, marginal endogenous tyrosine phosphorylation of SD-4 was observed [36] In addition, the data reported in these experiments indicate that tyrosine-phosphorylated SD-4 coassociates with tyrosine phosphorylated CXCR4, and suggest GAG to be independent of this association As the tyrosine phosphorylation of intact SD core proteins is not easily detected, we examined here the protein core of tyrosine phosphorylated SD-4 after digestion of the GAG chains with heparitinases I and III and chondroitinase ABC The 50–55 kDa proteins which were revealed with anti-SD-4 mAb 5G9 and with anti-Ptyr mAb 4G10 in the respective glycosaminidases-treated anti-Ptyr IP and anti-SD-4 IP probably represent dimers of tyrosine-phosphorylated SD-4 Similar apparent relative molecular masses of the SD-4 protein core were observed in the enriched PGs from glycosaminidases-treated cell lysates We then observed firstly an increase in SD-4 tyrosine phosphorylation, and secondly that homo- or hetero-oligomerization of CXCR4, induced by SDF-1 on HeLa cells, was prevented if the cells were pretreated with heparitinases I and III This indicates the involvement of HS in these two events In this study, in parallel experiments, either the cells were treated with heparitinases I and III or the IPs were treated with three glycosaminidases, heparitinases I and III and chondroitinase ABC To preserve cell viability, lower concentrations of heparitinases were used to treat the cells than the IPs According to these different conditions, GAGs, especially chondroitin sulfates, were still present on SD-4, if the enzyme treatment was performed on the cells This explains why incomplete deglycanation of SD-4 was observed if the cells were treated with heparitinases Finally, we asked whether HS and SD-4 were involved in other SDF-1-induced cellular activation signals As SDF-1 binding to CXCR4 activates p44 ⁄ p42 MAPK and JNK ⁄ SAP kinases and calcium mobilization [21,24–27], we compared the activation on either untreated or heparitinase I and III-treated HeLa cells In parallel, we investigated whether the reduction of expression of SD-4 on HeLa cells by the use of RNA interference prevented these activations HS removal from HeLa cells or decreasing endogenous FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 SD-4 significantly reduced the phosphorylations of p44 ⁄ p42 MAPK and JNK ⁄ SAP kinases induced by SDF-1 By contrast, these treatments did not change the calcium mobilization triggered by the chemokine These data indicate that HS and SD-4 are selectively required, at least partly and either directly or indirectly, for the activation of p44 ⁄ p42 MAPK and JNK ⁄ SAP kinases by SDF-1 on HeLa cells In conclusion, this study strongly suggests that 1-SD-4 behaves as an SDF-1 receptor selectively involved in transduction pathways induced by SDF-1 on HeLa cells and 2-HS play a role in these events Whether these observations correlate with a biological activity of SDF-1 deserves further study Experimental procedures Cell culture HeLa cells were cultured in DMEM (Invitrogen Corp., Paris, France) containing 10% fetal bovine serum (FBS; Biowhittaker, Paris, France) and l-glutamine (2 mm; Invitrogen Corp.), and split twice a week Flow cytometry Flow cytometry was performed as described [17,38,39], using anti-SD-1 mAb DL-101 (mouse IgG-1; clone DL-101; specific for the ectodomain of SD-1 of human origin), anti(SD-4) mAb 5G9 (mouse IgG2a; clone 5G9; specific for the ectodomain of SD-4 of human origin); anti-(SD-2) (goat IgG; specific for the C-terminal domain of syndecan-2 of human origin) (all from Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) or anti-(beta-glycan) Igs (goat IgG; R & D systems, Abingdon, UK), anti CD44 mAb (mouse IgG2a; Serotec, Oxford, UK), anti-CXCR4 mAb 12G5 (mouse IgG2a; specific for the second extracellular domain of CXCR4; BD Bioscience Pharmingen, San Diego, USA), or their isotypes (mouse IgG1, IgG2a or goat IgG, Jackson Immunoresearch, Laboratories Inc (Baltimore, MD, USA) or BD Bioscience Pharmingen (San Diego, CA, USA), all at 10 lgỈmL)1 Preparation of PGs The PGs from HeLa cells lysates were enriched by anion exchange chromatographies, as described previously [38] Binding of biotinylated SDF-1 to electroblotted PGs Enriched PGs were loaded onto 12% SDS ⁄ polyacrylamide gels (Invitrogen Corp.) under non reducing conditions and blotted onto polyvinylidene difluoride membranes 1947 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 (Amersham Pharmacia Biotech., Little Chalfont, Bucks, UK) as described [39] After blocking, strips were incubated for h at room temperature with biotinylated SDF-1a (6.25 nm; synthesized by F Baleux, Institut Pasteur, Paris, France; it was verified that biotin incorporation did not modify the chemokine behavior) After washing, strips were reacted with streptavidin-peroxidase (1.5 lgỈmL)1, Sigma-Aldrich, St Louis, MO, USA) for 60 at room temperature and revealed by enhanced chemoluminescence (ECL) detection (Amersham Pharmacia Biotech; or Supersignal West Dura ` Extended, Pierce, Perbio Science, Brebieres, France) Alternatively, strips were incubated for h at room temperature with anti-(SD-1) DL-101, anti-(SD-4) 5G9, anti-HS 10E4 or 3G10 mAbs (the latter two from Seikagaku, Tokyo, Japan), anti-SD-2 or anti-beta-glycan Abs or their isotypes (mouse IgG1, IgG2a, IgM, IgG2b or goat IgG) After washing, strips were incubated with HRP-labeled antimouse Ig (dilution of : 5000; Amersham Pharmacia Biotech) and developed In some experiments, before the binding assay, electroblotted PGs were digested for 18 h at 37 °C with 10 mmL)1 heparitinase III (heparin lyase; EC 4.2.2.7), 20 mmL)1 heparitinase I (heparan sulfate lyase; EC 4.2.2.8) and 33 mmL)1 chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4) (all from Sigma–Aldrich) as described previously [39] Immunofluorescence staining and confocal microscopic analysis of the cells To determine whether SDF-1 colocalizes with SD-4, HeLa cells were incubated with anti-(SD-4) mAb 5G9, which was revealed by Cy-3 donkey anti-mouse Igs (1 : 400; Jackson Immunoresearch, West Grove, PA, USA) Cells were then subsequently incubated for h at °C with 1-biotinylated SDF-1a (10 lgỈmL)1) Cells were then labeled for 30 at °C with a streptavidin-Alexa Fluor 488 complex (1 : 100, Molecular Probe, Inc., Eugene, OR, USA) and fixed with paraformaldehyde (Sigma-Aldrich) As controls, cells were incubated with the isotypes or biotinylated SDF-1a was omitted Cells were mounted and observed using a Zeiss microscope (Axiovert 135 m; Carl Zeiss AG, Gottingen, ă Germany) in conjunction with a confocal laser scanning unit (Zeiss LSM 410) Immunoelectron microscopy The HeLa cells were grown until 80% confluence in multiwell chambers After washes with phosphate buffered saline (NaCl ⁄ Pi), cells were incubated for h at °C with anti(SD-4) mAb 5G9 (20 lgỈmL)1) or anti-(SD-1) mAb DL101 (20 lgỈmL)1), which was followed by an incubation for 30 at °C with a donkey anti-(mouse IgG) Ig linked to 6-nm colloidal gold particles (Aurion, AA Wageningen, the Netherlands) The cells were then incubated for h at 1948 N Charnaux et al °C with 1-biotinylated SDF-1a (20 lgỈmL)1), which was followed by an incubation with streptavidin linked to 15 nm colloidal gold particles (Aurion) Cells were then post-fixed with 2.5% (v ⁄ v) glutaraldehyde (Sigma-Aldrich), dehydrated in graded ethanol series, and embedded in epoxy resin Ultra-thin sections (100 nm) were performed and observed in transmission electron microscopy (CM-10, Philips Medical Systems, Suresne, France) at high magnification (· 27 500) Immunoprecipitation and western blot analysis HeLa cells were washed with NaCl ⁄ Pi and cultured for 48 h in DMEM supplemented with 0.1% (v ⁄ v) FBS and incubated for 0, 2, 10, 30 at 37 °C with SDF-1a (0 up to 125 nm) In some experiments, cells were pretreated for h at 37 °C with heparitinase I (0.1 mL)1) and heparitinase III (0.2 mL)1) mixture It was verified that these enzymes treatment had no effect on cell viability, as assessed by Trypan blue exclusion dye After washing the cells with NaCl ⁄ Pi supplemented with orthovanadate (1 mm, Sigma-Aldrich), whole-cell extracts were prepared by lysis of the cells in 20 mm Tris, 150 mm NaCl, mm orthovanadate, 1% (v ⁄ v) NP-40, 10 mm phenylmethylsulfonyl fluoride, mm iodoacetate, 25 mm phenanthrolin and 20 lgỈmL)1 aprotinin (all from Sigma-Aldrich), The protein concentration in whole-cell extracts was determined by the BCA protein assay (Pierce) These extracts were then supplemented with 10 mm dithiothreitol (Sigma-Aldrich) Thereafter, equal amounts of proteins from these extracts were incubated for 18 h at °C with 100 lL of Protein GSepharose beads (Amersham Pharmacia Biotech), precoated either by anti-Ptyr mAb 4G10 (mouse IgG2b; Upstate Biotechnology, Inc, Lake Placid, NY, USA), anti-SD-4 mAb 5G9, or anti-CXCR4 Abs G19 (goat IgG; specific for the first extracellular domain of CXCR4; Santa Cruz Biotechnology) (each at lg), as described previously [33,40,41] To release bound components, beads were then boiled for 10 with 300 lL of 2· sample buffer for SDS ⁄ PAGE and centrifuged (400 g; at 15 °C) Cell lysates, eluates or eluted proteins were submitted to 12% SDS ⁄ PAGE under non reducing conditions and then transferred onto polyvinylidene difluoride membranes Complexes were revealed by incubation for h at room temperature with either anti-SD-4 5G9, anti-Ptyr 4G10, anti-CXCR4 12G5, anti-HS 10E4 mAbs, anti-SD-2 Igs or anti-(beta-glycan) Igs or their isotypes (all at : 1000–1 : 2000) After washing, strips were incubated with HRP-labeled anti-(mouse Ig) (at : 5000) and revealed by ECL reagent In some experiments, the immunocomplexes immobilized on the beads were treated by heparitinase I (1 mL)1), heparitinase III (15 mL)1) and chondroitinase ABC (5 mL)1) mixture Beads were washed Bound components were then eluted as just described and then electroblotted Results were quantified by scanning the exposed X-ray film with an Agfa scanner FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS N Charnaux et al and analyzed using an area measurement from scion imager They were expressed as the ratios of the data observed for the SDF-1 stimulated cells relative to those observed for the corresponding untreated cells The significance of the differences was assessed with a t-test Activation of p44/p42 MAPK and JNK/ SAP kinases by SDF-1 HeLa cells were washed with NaCl ⁄ Pi and cultured for 48 h in DMEM supplemented with 0.1% (v ⁄ v) FBS In some experiments, cells were pretreated for h at 37 °C with heparitinases I and III mixture, as just described It was verified that these enzymes treatment had no effect on cell viability, as assessed by Trypan blue exclusion dye Thereafter, cells were incubated for 0–30 at 37 °C with SDF-1a (at 0–125 nm) After washing with NaCl ⁄ Pi-orthovanadate (1 mm), whole cell extracts were prepared [39] The amount of protein of these extracts was controlled by using a protein detection kit (Pierce) Equal amounts of total proteins from these extracts were then submitted to 10% SDS ⁄ PAGE and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech) MAPKs were detected using polyclonal Abs, respectively, specific for phospho-p44 ⁄ p42 [Thr202 ⁄ Tyr204], phospho-SAPK ⁄ JNK [Thr183 ⁄ Tyr185], total p44 ⁄ p42 or total SAPK-JNK (rabbit IgG; all from Cell Signaling Technology) Revelation was performed as described [39] Quantification of p44 ⁄ p42 MAPKs- and of SAPK ⁄ JNK phosphorylations was performed by using the scion imager after autoradiography scanning For each sample, data were expressed as a ratio of p44 ⁄ p42 MAPKs- or SAPK ⁄ JNK-phosphorylated proteins over total proteins, in absorbance units The mean ± SE of triplicate determinations of individuals experiments was calculated and the statistical significance of the differences was evaluated using the Student’s t-test RNA interference SD-4 gene-specific sense and antisense 21 nt single-stranded RNAs (ssRNAs) with symmetric nt-3¢(2¢-deoxy) thymidine overhangs, were chemically synthesized, HPLC purified (Eurogentec, Seraing, Belgium) and used RNA sequences corresponding to SD-4 double strand RNA (SD-4 dsRNA) were: sense 5¢-GUU-GUC-CAU-CCC-UUG-GUGCdTdT-3¢; antisense 5¢-GCA-CCA-AGG-GAU-GGA-CAACdTdT-3¢ To verify the sequence specificity of the RNA interference, a SD-4 double-stranded RNA with one mismatch (mutSD-4 dsRNA) was used as negative control as described [28,29]: sense 5¢-GUU-GUC-GAU-CCCUUG-GUG-CdTdT-3¢; antisense 5¢-GCA-CCA-AGG-GAUCGA-CAA-CdTdT-3¢ For RNA interference experiments, double-stranded RNAs were generated by mixing equimolar amounts (50 lm) of sense and antisense ssRNAs in annealing buffer (50 mm Tris, pH 7.5–8.0, 100 mm NaCl in FEBS Journal 272 (2005) 1937–1951 ª 2005 FEBS Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 DEPC-treated water) for at 94 °C, followed by 60-min incubation at 37 °C HeLa cells were tranfected with 300 nm dsRNA in serum-free medium using Jetsi tranfectant reagent (Eurogentec) following the manufacturer’s instructions Mock cells were cultured in parallel and transfected with the transfection mixture lacking dsRNA Cells transfected with SD-4 dsRNA or mutSD-4 dsRNA were used days posttranfection for further analysis The efficiency of the RNA interference experiments was assayed by analyzing the respective expressions of the mRNAs from SD-4, SD-2, SD-1 and CXCR4 In parallel, the protein expressions of SD-4, SD-1, SD-2, beta-glycan, CXCR4 were analyzed by indirect immunofluorescence and FACS analysis SD-4 mRNA, SD-1 mRNA, SD-2 mRNA and CXCR4 mRNA and, to normalize for input of total RNA, glyceraldehyde 3-phosphodehydrogenase (GAPDH) mRNA were quantified by RT-PCR Total cellular RNA was extracted, using a Qiagen RNA ⁄ DNA Mini Kit (Qiagen S.A., Cortaboeuf, France) For this purpose, confluent monolayers of mocktransfected HeLa cells, mutSD-4 dsRNA-transfected HeLa cells and from SD-4 dsRNA transfected HeLa cells were previously grown in a six-well tissue culture Reverse transcription was performed using a Advantage RT-for-PCR Kit (BD Biosciences Clontech, Le Pont-de-Claix, France) The following synthetic SD-4 primers were used: – upper primer CGA 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 SD4 The following SD-1 primers were used: sense primer, 5¢-TCTGACAACTTCTCCGGCTC-3¢; antisense primer: 5¢-CCACTTCTGGCAGGACTACA-3¢; these primers were designed to amplify a 211 bp coding sequence of SD-1 The following synthetic SD-2 primers were used: sense primer 5¢-GGGAGCTGATGAGGATGTAG-3¢; antisense primer 5¢-CACTGGATGGTTTGCGTTCT-3¢ These primers were designed to amplify a 394 bp coding sequence of SD-2 The following synthetic CXCR4 primers were used: sense primer: 5¢-ATCTTTGCCAACGTCAGT-3¢; antisense primer: 5¢-TCACACCCTTGCTTGATG-3¢ These primers were designed to amplify a 308 bp coding sequence of CXCR-4 Optimum semiquantitative RT-PCR conditions were established to remain within the exponential phase of amplification curve After 23 cycles of amplification, 30 lL were electrophoresed in 2% agarose and analyzed Intracellular Ca2+ mobilization Possible changes in intracellular calcium concentration were monitored using the fluorescent probe Fluo-3 ⁄ AM (Molecular Probes) HeLa cells were washed in phenol redand sodium bicarbonate-free RPMI 1640 (Invitrogen Corporation), supplemented by 25 mm Hepes (Sigma-Aldrich) They were then incubated in the dark for 30 at 37 °C, with lm Fluo-3 acetoxymethyl ester (Fluo-3 ⁄ AM) which 1949 Syndecan-4 is an auxiliary receptor for SDF-1/CXCL12 has been previously solubilized in dimethylsulfoxide (Sigma-Aldrich), supplemented by Pluronic F-127 (20%) (Molecular Probes) Cells were then washed with RPMI 1640 and maintained in this buffer at 20 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