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 su
Trang 1factor-1 (SDF-1)/ CXCL12
Nathalie Charnaux1,2*, Se´verine Brule1,2*, Morgan Hamon1, Thomas Chaigneau1, Line Saffar1, Catherine Prost1, Nicole Lievre1and Liliane Gattegno1,2
1 Laboratoire de Biologie Cellulaire, Biothe´rapies Be´ne´fices et Risques, UPRES 3410 Universite´ Paris XIII, Bobigny, France
2 Hoˆpital Jean Verdier, Bondy, France
Chemokines are low molecular mass proteins mediating
several functions such as hematopoiesis regulation,
leu-kocyte 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
Keywords
CXCR4; proteoglycan; SDF-1⁄ CXCL12;
syndecan-4
Correspondence
L Gattegno, Laboratoire de Biologie
Cellulaire, Biothe´rapies Be´ne´fices et
Risques, UPRES 3410 Universite´ Paris XIII,
74, rue Marcel Cachin, 93017, Bobigny,
France, Hoˆpital Jean Verdier, 93017, Bondy,
France
Fax: +33 1 48026503
Tel: +33 1 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 CXCL12-independent 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-phos-phorylated 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 hepari-tinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44⁄ p42 mitogen-activated protein kinase and Jun N-ter-minal⁄ stress-activated protein kinase activation by action of CXCL12 on HeLa cells However, these treatments did not modify the calcium mobil-ization 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
Abbreviations
dsRNA, double-stranded RNA; FBS, fetal bovine serum; GAG, glycosaminoglycan; HS, heparan sulfate; JNK ⁄ SAPK, Jun N-terminal ⁄ stress-activated protein kinase; MAPK, mitogen-stress-activated-protein kinase; PFA, paraformaldehyde; PMA, phorbol 12-myristate-13-acetate; PG, proteoglycan; Ptyr, tyrosine phosphorylated; SDF, stromal cell-derived factor; SD, syndecan.
Trang 2embryonic 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
dis-tinct cell-, tissue-, and development-specific patterns
[12], and show cell-specific variations in the structure of
their HS chains [13] SDs may regulate
ligand-depend-ent 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
lympho-cytes and macrophages, which comprise CXCR4, as
expected, and also SD-4 [17], but not SD-1, SD-2,
beta-glycan or CD44 ([17] and unpublished data) Moreover,
we recently demonstrated the occurrence of an
SDF-1-independent heteromeric complex on the plasma
mem-brane 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
How-ever, our previous data have shown that while
glycos-aminidases pretreatment of primary macrophages
decreases the binding of SDF-1 to CXCR4, such
treat-ment 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
phos-phorylation 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,
tyrosine-phosphorylated SD-4 is physically coassociated with
tyrosine-phosphorylated CXCR4, and what the
GAG-dependency of these events is Finally, we asked
whe-ther SD-4 is involved in owhe-ther 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
path-ways induced by SDF-1 on these cells
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
B
c
Fig 1 PGs on HeLa cells (A) Cell surface expression of SD-1, SD-4 and CD44 on HeLa cells HeLa cells (5 · 10 5
) 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 2 · 10 6
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 (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.
Trang 3labeling 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
immunoreac-tive with both anti-SD-2 mAbs and mAb 3G10,
45- and 90 kDa proteins immunoreactive with
anti-SD-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
iso-types (data not shown) The fact that all these PGs
were also immunoreactive with anti-HS mAb 10E4,
but not with its isotype, demonstrates their glycana-tion (Fig 2, lane 6 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 7 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
8 vs 6), and strongly decreased that of biotinylated SDF-1a to SD-4 (Fig 2, lane 9 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 polyvinylid-ene difluoride membrane (data not shown); and (c) the direct binding of SDF-1 to SD-4 was GAG dependent
Confocal microscopy analysis showed that fluores-cently labeled biotinylated SDF-1a colocalizes with SD-4 on the plasma membranes of these cells, as assessed by the yellow (red-green colocalization) stain-ing (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 labe-led 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 2 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 represen-tative of three individual experiments.
Trang 4SDF-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 2 vs 1, and lane 4 vs 3)
These increases were not significant if the cells were
stimulated with 3 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 2 min or 30 min in the presence of
125 nm SDF-1 and were strongly significant after
10 min of incubation of the cells with 125 nm of the
chemokine (Fig 4A and data not shown) Among
these tyrosine phosphorylated proteins, those
immuno-reactive with anti-CXCR4 mAb 12G5 probably
repre-sent, respectively, CXCR4 monomers and homo- or
hetero-oligomers (Fig 4A, lane 4) Residual
phos-phorylation of CXCR4 in unstimulated cells was
detected (Fig 4A, lanes 1 and 3), as reported previously [21–23]
SDF-1 also induces the tyrosine phosphorylation
of SD-4 on HeLa cells and tyrosine phosphory-lated 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 des-cribed, was also characterized by a 110–200 kDa broad smear, which was marginally revealed if the cells were not stimulated (Fig 4A, lane 2 vs 1) and was not detected if the anti-Ptyr 4G10 IP was revealed with anti-CXCR4 mAb 12G5 (Fig 4A, lane 4 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-unactivated-and SDF-1-activated HeLa cell lysates were immuno-precipitated in parallel with anti-Ptyr mAb 4G10 and blots were developed with several different anti-PG Abs: 4 mAb 5G9, 1 mAb DL-101,
anti-SD-2 Abs or anti-beta-glycan Abs (Fig 4B and data not shown) The tyrosine phosphorylated smear described above was only significantly observed when the anti-Ptyr 4G10 IP from SDF-1-activated HeLa cell lysates
Syndecan-4 SDF-1α Merged
A
B
Fig 3 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 experi-ments Bar ¼ 5 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
6 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 rep-resentative of three individual experiments.
Trang 5was revealed with anti-SD-4 mAb 5G9 (Fig 4B, lane 3
vs 6, 8 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
concentra-tion-dependent: it was marginal if the cells were
incuba-ted for 2 min or 30 min with 3, 50 or 125 nm of SDF-1,
and significant if the cells were incubated for 10 min
with 125 nm SDF-1 (Fig 4B, lanes 3 vs 2, 4 and data
not shown) These latter conditions were therefore used
for the following IPs To further demonstrate the
occur-rence of tyrosine-phosphorylated SD-4, the
SDF-1-unactivated- and SDF-1-activated HeLa cell lysates
were precipitated with anti-SD-4 mAb 5G9 and
devel-oped with anti-Ptyr mAb 4G10 (Fig 5A, lanes 1 and 2)
To confirm equal loading of the samples, the 5G9 IPs
were stripped and reprobed with anti-SD-4 mAb 5G9 (Fig 5A, lanes 5 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 phos-phorylated 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-unstimu-lated and stimuSDF-1-unstimu-lated HeLa cells were treated with a
D C
Fig 4 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 equival-ent 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 min with 125 n M 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 n M 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 phos-phorylated bands shown in A and B (lanes 1–4) were quantified in absorbance units by densitometric scanning and analyzed with SCION
IMAG-ER 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 Pro-tein bands with changes in tyrosine phosphorylation state are indicated by arrows.
Trang 6A
C
Fig 5 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 2 and 4) or not (–) (lanes 1 and 3) for 10 min with 125 n M SDF-1a In some experiments, cells were pretreated in parallel with heparitinases I and III mixture (lanes 3 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 5 and 6 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 stimula-ted (+) (lanes 2, 4, 6 and 8) or not (–) (lanes 1, 3, 5 and 7) for 10 min with 125 n M SDF-1a Cells lysates were immunoprecipitated with anti-Ptyr 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 1 and 2), anti-(SD-1) DL-101 mAb (lanes 3 and 4), anti-Ptyr 4G10 mAb (lanes 5 and 6) or the isotype IgG2b (lanes 7 and 8) (C) Upper panel: HeLa cells were stimulated (+) (lanes 2 and 3) or not (–) (lane 1) for 10 min with 125 n M 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 correspond-ing 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.
Trang 7mixture 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
Pro-teins 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-(beta-glycan) 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 1 and 2) Therefore, the
SDF-1-independent heteromeric complex between CXCR4 and
SD-4 (Fig 5C, lane 1) is still present if the cells are
sti-mulated by the chemokine (Fig 5C, lane 2 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
pre-serve 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
incu-bated 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
hepari-tinases I and III, this smear was no longer present
(data not shown) Moreover, this heparitinases
pre-treatment 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 4 vs 3) In
these experiments, the apparent relative molecular
masses of most tyrosine-phosphorylated SD-4
mole-cules were also decreased, as expected [17] (Fig 5A,
lanes 3 and 4 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-
oligo-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 3
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-phosphoryl-ated SD-4 with tyrosine phosphoryltyrosine-phosphoryl-ated 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 interac-tions are not involved in these physical associainterac-tions Finally, in all the experiments described above, results of immunoprecipitation of cell lysates with iso-type-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 elec-troblotted and revealed using phospho-specific anti-p44⁄ 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-myristate-13-acetate (PMA) induced a rapid activation of p44⁄ 42 MAPK and JNK⁄ SAP kinase signaling in HeLa cells
by increasing phosphorylations of the respective pro-teins (Fig 6) This effect was time and concentration-dependent: It rose from 3 nm up to 125 nm SDF-1a and if the time of incubation with the chemokine was enhanced from 5 to 15 min On the contrary, these phosphorylations decreased if the time of incubation with the chemokine was enhanced up to 30 min (Fig 6A) According to these results, the cells were incubated for 15 min 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)
Trang 8As expected [21,26,27], SDF-1 also stimulates
intra-cellular 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 ±
SE¼ 101 ± 21, n ¼ 30) as compared to untreated
control cells (mean ± SE¼ 97 ± 16, n ¼ 32), or the
percentage of SDF-1 responding cells (Fig 7A,B and
data not shown)
Transfection of HeLa cells with SD-4 double-stran-ded RNA (SD-4 dsRNA) resulted, as expected, in a SD-4 mRNA downregulation reaching 80% reduc-tion 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
400
300
200
100
300
200
100
0
A
B
Fig 6 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 3 n M or 125 n M 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 min with PMA (0.5 l M ) or SDF-1a (125 n M ).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 (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 phos-phorylated proteins over total proteins in absorbance units The amount of MAPK (p44 ⁄ p42 or SAPK ⁄ JNK) phosphorylation in the SDF-1-sti-mulated cells was calculated according to the level of phosphorylated MAPK proteins in unstiSDF-1-sti-mulated 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.
Trang 9(Fig 8B–D and data not shown) To monitor
the sequence specificity for SD-4 RNA interference,
4 dsRNAs was used as a control The
mutSD-4 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) (P¼ 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
stimula-tion, as compared with the data observed in
mock-transfected cells and in cells mock-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,
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 bind-ing 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 selec-tivity 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 electroblot-ted 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 do 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 cyto-plasmic and transmembrane domains, in contrast their
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heparitinases I and III treated HeLa cell untreated HeLa cell
SD-4 dsRNA HeLa cell mock-transfected
HeLa cell
Fig 7 Heparan sulfate is not involved in
int-racellular Ca 2+ mobilization induced by
SDF-1 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 min with Fluo-3 and then
stimulated with SDF-1a (125 n M ), as
indica-ted 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.
Trang 10extracellular 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
tyro-sine residues, two of which are in favorable sequences
for phosphorylation [36] Endogenous tyrosine phos-phorylation of SDs has already been detected while most cell surface SDs are phosphorylated following treatment with the tyrosine phosphatase inhibitor per-vanadate [37] Tyrosine phosphorylation of the SD cytoplasmic domain may be a common mechanism for regulating SD activity In this study, immunoprecipita-tion experiments using Ptyr, CXCR4 and anti-(SD-4) mAbs show for the first time that besides the tyrosine phosphorylation of CXCR4, tyrosine phos-phorylation of SD-4 occurs in response to SDF-1 sti-mulation of HeLa cells This tyrosine phosphorylation depends on the time of incubation of the cells with the chemokine: marginal for 2-min incubation, significant
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(SD-4ds RNA)
(mocktransfected)
Fig 8 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,
3 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 rep-resents the mean ± SE of triplicate determination of an individual experiment The significance of the differences as compared to mock-transfected 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, 3 days post transfection Reactivity was compared to an isotype-matched control mAb (E) Upper panel: HeLa cells were treated for 15 min with 125 n M SDF-1a, 3 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 rab-bit 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.