Upload
lytu
View
221
Download
0
Embed Size (px)
Citation preview
Supplementary Information
Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors
Florian Mullershausen1¶, Frédéric Zecri2, Cihan Cetin4, Andreas Billich3, Danilo Guerini3 and Klaus Seuwen1¶
Novartis Institutes for BioMedical Research, 1Developmental & Molecular Pathways, 2Global Discovery Chemistry, 3Autoimmunity, Transplantation & Inflammation, Novartis Pharma AG, Basel, Switzerland. 4Friedrich Miescher Institute for Biomedical Research, Facility for Advanced Microscopy and Imaging, Basel, Switzerland ¶To whom correspondence should be addressed: Florian Mullershausen or Klaus Seuwen Novartis Pharma AG Forum 1 Novartis Campus CH-4056 Basel Tel. +41 61 324 2910 Fax. +41 61 324 6105 [email protected]@novartis.com
1Nature Chemical Biology: doi: 10.1038/nchembio.173
Supplementary Figures
a
0
S1P
FTY720P
0
25
50
75
100
0.001 0.01 0.1 1 10 100[Agonist] (nM)
cAM
P (%
of F
sk)
- FTYP S1P
Fsk+PTX
cAM
P (%
of F
sk)
0
25
50
75
100
125
b Fsk
S1P S1P- FTYP
FTYP FTYP- FTYP
- -- FTYP
0
20
40
60
80
100
120
cAM
P (%
of F
sk)
Preinc.:Assay:
a
0
S1PS1P
FTY720P
0
25
50
75
100
0.001 0.01 0.1 1 10 100[Agonist] (nM)
cAM
P (%
of F
sk)
- FTYP S1P
Fsk+PTX
cAM
P (%
of F
sk)
0
25
50
75
100
125
b Fsk
S1P S1P- FTYP
FTYP FTYP- FTYP
- -- FTYP
0
20
40
60
80
100
120
cAM
P (%
of F
sk)
Preinc.:Assay:
Fsk
S1P S1P- FTYP
FTYP FTYP- FTYP
- -- FTYP
0
20
40
60
80
100
120
cAM
P (%
of F
sk)
Preinc.:Assay:
Supplementary Figure 1: Control experiments using CHO-S1P1 and CHO-S1P3 cells (a) The inhibition of Fsk-induced formation of cAMP was measured in CHO-S1P1 cells in
the presence of the indicated concentrations of S1P or FTY720P. The bar graph shows
the responses to Fsk in PTX-treated cells in the absence or presence of maximally
effective concentrations of S1P or FTY720P, shown is a representative experiment. (b)
CHO-S1P3 cells were preincubated without or with FTY720P (1 µM) or S1P (1 µM) for 1
h, washed, and further incubated in the absence of agonists for 5 h. Then, Fsk-induced
cAMP formation was assayed in the absence of presence of FTY720P (10 nM). Data
represent mean ± SEM of ≥ 3 independent experiments.
2Nature Chemical Biology: doi: 10.1038/nchembio.173
40
60
80
100
120
0.1 1 10 100 1000
FTYP (nM) during preinc.
cAM
P (%
of F
sk)
5h washout24h washout
48h washout
40
60
80
100
120
0.1 1 10 100 1000
FTYP (nM) during preinc.
cAM
P (%
of F
sk)
5h washout24h washout
48h washout
a
FTYP FTYP10 nM 1 µM
control
PTX
Preinc.: --
0
100
200
300
cAM
P (%
of F
sk c
ontro
l)
bFsk
Supplementary Figure 2: Reversibility of persistent signaling and Gi-protein-mediated sensitization of cAMP formation (a) Fsk-induced cAMP formation in CHO-S1P1 cells that were preincubated without or
with FTY720P at the indicated concentrations for 1 h, washed, and further incubated in
the absence of agonists for 5, 24 or 48 h. (b) CHO-S1P1 cells were preincubated without
or with the indicated concentrations of FTY720P (1 h), washed and further incubated for
5 h with or without PTX in the absence of agonists. Then, Fsk-induced cAMP formation
was determined. Data represent mean ± SEM of ≥ 3 independent experiments.
3Nature Chemical Biology: doi: 10.1038/nchembio.173
a
b
60
Cel
l cou
nt
100 101 102 103
20
40
0
FTY-P
OC6
OC5
AFD-R control
Fluorescence
c
0
5
10
15
20
FTYP AFD-R OC6 OC5 control
Ave
rage
Dis
tanc
e
0
20
40
60
80
100
FTYP AFD-R OC6 OC5 control
Sur
face
exp
ress
ion
(% o
f con
trol)
a
b
60
Cel
l cou
nt
100 101 102 103
20
40
0
FTY-P
OC6
OC5
AFD-R control
Fluorescence
c
0
5
10
15
20
FTYP AFD-R OC6 OC5 control
Ave
rage
Dis
tanc
e
0
20
40
60
80
100
FTYP AFD-R OC6 OC5 control
Sur
face
exp
ress
ion
(% o
f con
trol)
Supplementary Figure 3: Quantification of S1P1 receptor distribution in CHO-S1P1 cells (a) CHO-S1P1 cells expressing an N-terminal extracellular myc-epitope tag were
stimulated with the indicated agonist (1 µM, 1h) and stained with an anti-myc antibody
and a secondary antibody labeled with Alexa 488 fluorescent dye. 10,000 viable cells in
each sample were analyzed using flow cytometry. (b) Cell surface localization of S1P1
as detected by flow cytometry expressed as geometric mean ± SEM derived from ≥ 3
independent experiments. (c) Images of CHO-S1P1 cells treated with the indicated
agonists (1 µM, 1h) and stained with anti-S1P1 antibodies were analyzed as described
in the Supplementary Methods section. Data are expressed as average Mahalanobis
Distance from FTYP-treated cells and were derived from 40 – 60 single cells.
4Nature Chemical Biology: doi: 10.1038/nchembio.173
a
W146 - -
FTYP(1 nM)
FTYP(10 nM)
S1P(100 nM)
- + - + - +
control
Fsk
0
20
40
60
80
100
120
cAM
P(%
Fsk
)
a
W146 - -
FTYP(1 nM)
FTYP(10 nM)
S1P(100 nM)
- + - + - +
control
Fsk
0
20
40
60
80
100
120
cAM
P(%
Fsk
)
b
W146:
pERK
Calnexin
S1P (nM)FTYP (nM)
0
-
1
-
1
+
10
-
10
+
100
-
100
+
b
W146:
pERK
Calnexin
S1P (nM)FTYP (nM)
0
-
1
-
1
+
10
-
10
+
100
-
100
+
0
25
50
75
100
125c
FTYP FTYPPTX
Preinc.: -
Fsk
cAM
P(%
Fsk
)
0
25
50
75
100
125c
FTYP FTYPPTX
Preinc.: -
Fsk
cAM
P(%
Fsk
)
Supplementary Figure 4: Characterization of S1P receptor-mediated signal transduction in HUVECs (a) Inhibition of Fsk-induced cAMP formation in HUVECs in the presence of the indicated
concentrations of FTY720P or S1P. Where indicated, the antagonist W146 (10 µM) was
added to the cells 15 min prior to the assay. (b) Western blot detection of pERK after
stimulation of HUVECs with the indicated concentrations of FTY720P or S1P. Where
indicated, W146 (10 µM) was added to the cells 25 min prior to stimulation. Calnexin
was detected to control for protein content of the samples. (c) HUVECs were
preincubated without or with the indicated FTY720P (1 µM, 1 h), washed and further
incubated for 4 h with or without PTX in the absence of agonists. Then, Fsk-induced
cAMP formation was determined.
5Nature Chemical Biology: doi: 10.1038/nchembio.173
0
25
50
75
100
125
S1P S1P- FTYP
FTYP FTYP- FTYP
- -- FTYP
Preinc.:Assay:
cAM
P(%
Fsk
)
Fsk
0
25
50
75
100
125
S1P S1P- FTYP
FTYP FTYP- FTYP
- -- FTYP
Preinc.:Assay:
cAM
P(%
Fsk
)
Fsk Supplementary Figure 5: Persistent signaling of FTY720P in primary astrocytes Primary rat astrocytes were preincubated without or with FTY720P (1 µM) or S1P (1 µM)
for 1 h, washed, and further incubated in the absence of agonists for 5 h. Then, Fsk-
induced cAMP formation was assayed in the absence of presence of FTY720P (10 nM).
6Nature Chemical Biology: doi: 10.1038/nchembio.173
GM130 LAMP1
1 h
3 h
6 h
8 h
Supplementary Figure 6: Localization of S1P1 in HUVEC after long-term incubation with FTY720P Co-immunostaining of S1P1 receptors (green) and the indicated organelle markers (red)
in HUVECs stimulated with FTY720P (1 µM) for 1, 3, 6 or 8 h. Left panel shows GM130
(Golgi matrix), right panel shows LAMP1 (lysosomes). Scale bar: 10 µm
7Nature Chemical Biology: doi: 10.1038/nchembio.173
Supplementary Methods
Antibodies and Reagents
S1P was purchased from Biomol (USA) and dissolved in DMSO containing 50 mM HCl
to yield a stock solution of 5 mM. Rabbit polyclonal antibodies to human S1P1 receptor
were from Affinity BioReagents (USA) and from Santa Cruz Biotechnology (USA).
Mouse monoclonal antibodies against organelle resident antigens EEA1, CD107a,
GM130, p230 were from BD Biosciences (USA), mouse monoclonal antibodies against
clathrin and Mannose-6-phosphate receptor were from Abcam (UK). For Western blot
detection, antibody against phosphorylated ERK was from Cell Signaling (USA),
Calnexin antibody was from BD Biosciences (USA) and βActin antibody was from Sigma
(Switzerland).
In vitro phosphorylation of FTY720 and OC5
The phosphorylation reactions were performed essentially as described1. Briefly, the
cytoplasmic fraction of recombinant HEK-293 cells overexpressing human SPHK-2 was
incubated at 30°C in a total volume of 100 μL with FTY720 or OC5 (20 μM; added from
stock solutions in DMSO), 1 mM of ATP, and 2 μCi [γ-32P]ATP in 50 mM Hepes buffer
(pH 7.4) containing 15 mM MgCl2, 0.005% Triton X-100, 10 mM KCl, 10 mM NaF and
1.5 mM semicarbazide. Following incubation for different time points up to 2 h, lipids
were extracted and separated by thin-layer chromotography. Radiolabeled FTY720P
derivatives were visualized and quantified using a Molecular Dynamics Storm
PhosphorImager (Sunnyvale, CA). From the data, the rate of phosphorylation was
calculated as nmoles of product formed per min per mg of total protein.
8Nature Chemical Biology: doi: 10.1038/nchembio.173
Flow cytometric analysis of surface expression of S1P1 upon agonist treatment
CHO-S1P1 cells were grown in six-well plates to 60-80% confluence in αMEM, 10 %
charcoal-stripped lipid-depleted FBS, 50 µg/ml Gentamicin. The cells were incubated for
1h with a 1 μM agonist solution at 37°C in a CO2 incubator in complete medium. After
the incubation and treatment with the compounds the cells were washed with
phosphate-buffered saline (PBS) and detached by treatment with PBS/1 mM EDTA. 106
cells were incubated either with 4 μg/ml of monoclonal mouse anti c-myc IgG1 (Roche
Applied Science, Switzeraland) antibody or with isotype control mouse IgG1
(Pharmingen, BD Biosciences, Switzerland) in balanced salt solution (BSS)–5% FCS
(BSS: 0.14 M NaCl, 1 mM CaCl2, 5.4 mM KCl, 0.8 mM MgSO4, 0.3 mM NaH2PO4, and
0.4 mM KH2PO4 [pH 6.9]) for 60 min at 4ºC. The cells were washed once in BSS/5%
FCS, and incubated 60 min at 4ºC in the dark with 1 μg/ml of Alexa488-labeled goat anti
mouse secondary conjugates (Invitrogen, Switzerland). The cells were washed again
before being subjected to cytofluorometric analysis (FACS Calibur, BD Biosciences,
Switzerland). Flow cytometric measurements were performed with 10,000 viable cells
per sample. The viability was tested by the addition of 3 μl of a 1 μM DNA binding TO-
PRO-3 iodide solution (Invitrogen, Switzerland) and analyzed by flow cytometry.
Immunocytochemistry
Cells were seeded into 8-well chambered coverglasses (Lab-Tek II, NUNC, USA) and
grown for 2-3 days. 1 h prior to treatment, the growth medium was replaced by FCS-free
medium. After treatment, we fixed and permeabilized the cells using the
Cytofix/Cytoperm kit (BD Biosciences, USA). Chambers were washed with PBS,
Cytofix/Cytoperm solution was added (300 µl/well) and the slides were put on 4°C for 20
min. The wells were then rinsed twice with washing solution (1x, diluted from 10x stock;
BD Biosciences) and blocking buffer (140 mM NaCl, 10 mM Hepes, 5 mM CaCl2, 5%
9Nature Chemical Biology: doi: 10.1038/nchembio.173
FCS, 1% BSA, 0.04% NaN3 in washing solution) was added (300 µl/well) for at least 20
min at room temperature. Primary antibodies were diluted into blocking buffer and left on
the samples over night at 4°C. Cells were rinsed 2x and incubated with anti-rabbit and/or
anti mouse secondary antibodies labeled with Alexa 488 or Alexa 633 fluorescent dyes
(Invitrogen, Switzerland) for 2-3 h, washed and viewed with a Zeiss LSM 510 META
confocal microscope (Carl Zeiss AG, Germany) using 63x oil immersion (NA 1.4) or 63x
water immersion (NA 1.2) objectives; nuclei were stained with DAPI. Images were
acquired in multi-scan mode, i.e. all colors were scanned sequentially. In co-localization
studies, optical sections were recorded at a thickness of 1.2 µm for all color channels.
Image Analysis
Images were analyzed using the ImageJ software in order to quantify differences in
receptor localization after treatment of cells with the different compounds. Prior to the
analysis, a total of 40-60 cells per experimental condition was manually defined. A
customized plugin was implemented to analyse differences using Haralick texture
analysis2, Granulometry3 and Level-set4 methods. 22 features were measured for every
cell in every field of view, most of which were highly correlated. Principal Component
Analysis5 was used to uncorrelate the features obtained in the previous operation thus
reducing the number of dimensions from 22 to 13. Next, a covariance matrix of 13 non-
correlated features was generated based on all FTY720P-treated cells, which was then
used to calculate the Mahalanobis Distance6 of each single cell (of every treatment) from
the center of mass of FTY720P-treated cells. The Mahalanobis Distance has the
advantage of being scale-invariant compared to the Euclidian distance, so that features
with high values will not be weighed to a greater extent compared to a feature with low
values.
10Nature Chemical Biology: doi: 10.1038/nchembio.173
Western blot detection of phosphorylated ERK (pERK)
For Western blot detection of pERK, cells were cultured in 6-well plates. Starvation time
of cells in FCS-free medium was 4-5 hours prior to simulation. When indicated,
preincubations were carried out as described below. To induce ERK phosphorylation,
FTY720P was added at a final concentration of 10 nM and cells were incubated for 10
min at 37°C. Reactions were terminated by the addition of Laemmli sample buffer (Bio-
Rad, Switzerland) and boiling of the samples for 5 min. SDS-PAGE was carried out
using the Criterion system with 4-16% Tris-HCl gradient gels (Bio-Rad, Switzerland),
followed by electrotransfer of proteins onto nitrocellulose membranes. Incubation of the
membranes with primary antibodies was carried out over night at 4°C, for the control of
protein content of the samples antibodies against βActin (CHO-S1P1) or Calnexin
(HUVEC) were used. Appropriate secondary antibodies conjugated with horseradish
peroxidase (Pierce, USA) were applied followed by chemiluminescent detection of bands
using the SuperSignal™ West Dura detection kit (Pierce, USA). Visualization was
performed with a Chemidoc XRS system and the Quantity 1 software (Bio-Rad,
Switzerland).
Scratch-wound assay for migration
HUVECs were plated in 8-well µ-Slides with the ibiTreat surface (ibidi, Germany) and
grown to confluence. The growth medium was exchanged with basal endothelial medium
(without supplement, Promocell) supplemented with 1% charcoal-stripped FCS and
equilibrated for 4 h at 37°C. Pretreatments were carried out as above, by adding
FTY720P or S1P (1 µM) or control buffer and incubation of the cells for 60 min at 37°C.
Then, cells were washed 3x with PBS supplemented with CaCl2 (0.9 mM) and MgCl2
(0.5 mM). Cross-shaped scratches were introduced into each well using a 1000 µl sterile
plastic pipet tip (Eppendorf, Germany) and cells were washed again 2x to remove
11Nature Chemical Biology: doi: 10.1038/nchembio.173
debris. Cells were allowed to migrate in basal endothelial medium (1% charcoal-stripped
FCS) for 24 h. As a positive control, FTY720P (10 nM) was included in the medium
during the migration period. A picture of the same microscopic field was taken at the
beginning and the end of the migration period, using an Axiovert 200M microscope with
a 10x objective (Carl Zeiss AG, Germany). For analysis, the migration fronts were
defined and the distances were measured using the Zeiss LSM510 standard imaging
software (Carl Zeiss AG, Germany). The distance migrated was calculated by
subtracting the distance of the migration fronts measured at the beginning of the
experiment from the distance measured after the 24 h migration period. For each
experimental condition, 4 wells were used to obtain the mean value in every experiment.
12Nature Chemical Biology: doi: 10.1038/nchembio.173
Supplementary references
1 Billich, A. et al. Phosphorylation of the immunomodulatory drug FTY720 by
sphingosine kinases. J. Biol. Chem. 278, 47408-47415 (2003).
2 Haralick, R.M. Shanmugam, K. & Dinstein, I. Textural features for image
classification. IEEE Trans. on Systems, Man, and Cybernetics 3(6), 610–621
(1973).
2 Soille, P. Morphological Image Analysis: Principles and Applications, 2nd edition
(Springer Verlag, New York, 2003).
4 Osher, S. & Fedkiw, R. LSM: An overview and some recent results. UCLA IPAM
reports, 1-65 (2001).
5 Huang, K., Velliste, M. & Murphy, R. F. Feature Reduction for Improved Recognition
of Subcellular Location Patterns in Fluorescence Microscope Images. Proc. SPIE,
4962, 307-318. (2003)
6 Mahalanobis, P. On the generalized distance in statistics. Proc. Natl. Inst. Sci. India
2, 49-55 (1936).
13Nature Chemical Biology: doi: 10.1038/nchembio.173