Epithelial cell extrusion requires the sphingosine-1-phosphate receptor 2 pathway

Article (PDF Available)inThe Journal of Cell Biology 193(4):667-76 · May 2011with30 Reads
DOI: 10.1083/jcb.201010075 · Source: PubMed
To maintain an intact barrier, epithelia eliminate dying cells by extrusion. During extrusion, a cell destined for apoptosis signals its neighboring cells to form and contract a ring of actin and myosin, which squeezes the dying cell out of the epithelium. Here, we demonstrate that the signal produced by dying cells to initiate this process is sphingosine-1-phosphate (S1P). Decreasing S1P synthesis by inhibiting sphingosine kinase activity or by blocking extracellular S1P access to its receptor prevented apoptotic cell extrusion. Extracellular S1P activates extrusion by binding the S1P(2) receptor in the cells neighboring a dying cell, as S1P(2) knockdown in these cells or its loss in a zebrafish mutant disrupted cell extrusion. Because live cells can also be extruded, we predict that this S1P pathway may also be important for driving delamination of stem cells during differentiation or invasion of cancer cells.
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J. Cell Biol. Vol. 193 No. 4 667–676
JCB 667
JCB: Report
Correspondence to Jody Rosenblatt: jody.rosenblatt@hci.utah.edu
Abbreviations used in this paper: HBE, human bronchial epithelial; mil, miles
apart; S1P, sphingosine-1-phosphate; SKI, sphingosine kinase inhibitor; WT,
wild type.
Epithelia comprised of one or two cell layers cover and protect
the organs that they encase. The cells making up epithelia are
constantly turning over by cell division and apoptosis, yet cell
death could compromise the barrier function of the epithelium.
We previously found that epithelia use a process termed “apop-
totic cell extrusion” to remove apoptotic cells from a layer, while
preserving their barrier function (Rosenblatt et al., 2001). Speci-
cally, an early apoptotic epithelial cell triggers formation of an
actin and myosin ring in the live neighboring cells surrounding
it. Contraction of this ring then squeezes the dying cell out of the
epithelium. Apoptotic cell extrusion is conserved in all in vivo epi-
thelia we have examined ranging from Drosophila to human.
We previously showed that extrusion depends on a chemi-
cal signal from the apoptotic cells, which activates the Rho path-
way in the neighboring cells (Rosenblatt et al., 2001; Slattum
et al., 2009). Specically, addition of early apoptotic cells onto
an epithelial monolayer induces actin assembly in the live con-
tacted cells. Furthermore, inhibition of Rho in the cells sur-
rounding an apoptotic cell blocks extrusion (Rosenblatt et al.,
2001). We recently determined that Rho activation during extru-
sion requires p115 RhoGEF (Slattum et al., 2009), a protein
activated downstream of the G
G protein–coupled receptor
(Holinstat et al., 2003). Thus, a signal on the surface of the
dying cell triggers p115 RhoGEF to activate Rho-mediated
actin–myosin assembly and contraction in the live surrounding
cells to remove the dying cell. However, we did not know the
identity of the signal produced in early apoptotic cells that acti-
vates apoptotic cell extrusion.
Here, we report that the signal produced by dying cells is
the bioactive lipid sphingosine-1-phosphate (S1P), which acti-
vates actomyosin contraction in surrounding cells via the S1P
receptor. Inhibition of S1P synthesis or extracellular S1P sig-
naling blocks apoptotic cell extrusion. The cells surrounding the
dying cell require S1P
to bind S1P and activate formation
and contraction of the actomyosin-extruding ring in both tissue
culture and zebrash epithelia. Together, our data reveal the
signaling pathway that drives a cell to extrude from an epithe-
lial monolayer.
Results and discussion
Blocking S1P signaling inhibits extrusion of
apoptotic cells
To characterize the extracellular apoptotic signal that triggers
formation of the actin–myosin extruding ring, we used a modi-
ed version of our previous “cell addition assay.” In that assay,
o maintain an intact barrier, epithelia eliminate
dying cells by extrusion. During extrusion, a cell
destined for apoptosis signals its neighboring cells
to form and contract a ring of actin and myosin, which
squeezes the dying cell out of the epithelium. Here, we
demonstrate that the signal produced by dying cells to
initiate this process is sphingosine-1-phosphate (S1P).
Decreasing S1P synthesis by inhibiting sphingosine kinase
activity or by blocking extracellular S1P access to its
receptor prevented apoptotic cell extrusion. Extracellular
S1P activates extrusion by binding the S1P
receptor in
the cells neighboring a dying cell, as S1P
knockdown in
these cells or its loss in a zebrafish mutant disrupted cell
extrusion. Because live cells can also be extruded, we pre-
dict that this S1P pathway may also be important for driv-
ing delamination of stem cells during differentiation or
invasion of cancer cells.
Epithelial cell extrusion requires the sphingosine-1-
phosphate receptor 2 pathway
Yapeng Gu,
Tetyana Forostyan,
Roger Sabbadini,
and Jody Rosenblatt
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112
LPath, Inc. 6335 Ferris Square, Suite A, San Diego, CA 92121
© 2011 Gu et al. This article is distributed under the terms of an Attribution–Noncommercial–
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JCB • VOLUME 193 • NUMBER 4 • 2011 668
inhibitor of SphKs, all dramatically increased the percentage
of unextruded apoptotic cells compared with control treatment
(Fig. 1 F). Inhibition of apoptotic cell extrusion by SKIs was
dose dependent (Fig. S2, C–E). These results show that extru-
sion of apoptotic cells requires SphK activity, presumably be-
cause it catalyzes S1P synthesis.
To regulate many cellular responses, S1P is exported out
of cells and binds to its receptors on the same or neighboring cells.
To test whether extracellular S1P is necessary for apoptotic cell
extrusion, the MDCK monolayer was treated with UV in the
presence of an S1P-neutralizing mAb (Visentin et al., 2006;
O’Brien et al., 2009). Formation of the extrusion ring was inhib-
ited by the anti-S1P mAb, and consequently the apoptotic cell
failed to extrude and was instead retained in the monolayer (Fig. 2,
B and C). Additionally, the anti-S1P mAb also blocks actin ac-
cumulation induced by cell fragments (Fig. 2, E and F). These
results suggest that extracellular S1P is critical for extrusion.
Extrusion requires signaling through the
We next investigated which of the ve known high-afnity cell
surface S1P receptors (S1P
) might mediate extrusion signal-
ing. S1P
are ubiquitously expressed, whereas S1P
and S1P
are expressed mainly in lymphoid cells and neuronal cells, respec-
tively. By RT-PCR we determined that mRNAs from S1P
are expressed in MDCK cells and another epithelial cell line,
human bronchial epithelial (HBE) cells (unpublished data). To
investigate the involvement of S1P receptors in extrusion, we
treated an HBE monolayer with UV to induce apoptosis in con-
junction with several antagonists that are specic for different
S1P receptors, and evaluated extrusion of apoptotic cells. Nei-
ther FTY720-P (up to 10 µM), a functional antagonist of all S1P
receptors except S1P
(Brinkmann et al., 2002; Mandala et al.,
2002), nor VPC-23019 (up to 10 µM), an antagonist specic for
and S1P
(Davis et al., 2005), notably affected extrusion of
apoptotic cells (Fig. 3 A). In contrast, an S1P
-specic antago-
nist, JTE-013 (Osada et al., 2002), increased the proportion of
un-extruded apoptotic cells by approximately threefold (Fig. 3 A).
We obtained similar results with these S1P inhibitors in MDCK
monolayers (not depicted). These results suggested that S1P
signaling through S1P
, but not the other S1P receptors, is nec-
essary for cell extrusion.
To conrm the involvement of S1P
in extrusion and to
determine which cells require this receptor, we used shRNA to
silence S1P
expression in HBE cells. GFP-positive cells were
FAC-sorted and knockdown of S1P
was conrmed by quantita-
tive RT-PCR (Fig. 3 B). Knockdown of S1P
dramatically in-
creased the proportion of nonextruded apoptotic cells, compared
with control shRNA knockdown cells (Fig. 3, C and D). To con-
trol for off-target effects of hairpins, we used two additional
shRNA sequences to knock down S1P
and obtained similar re-
sults (Fig. S3, A and B). To test which cells require S1P
, we
examined extrusion in monolayers that were mosaically knocked
down for S1P
. A dying cell with S1P
knockdown surrounded
by cells with wild-type (WT) levels of S1P
extrudes success-
fully (Fig. 3 E, representing nine cases in three independent ex-
periments). By contrast, complete S1P
knockdown of all cells
addition of early apoptotic cells, but not late apoptotic cells or
live cells, to an intact Madin Darby canine kidney (MDCK) epi-
thelial monolayer induced actin assembly in the contacted living
cells. Necrotic cells likely use the same signal that apoptotic
cells use to produce an extruding ring in epithelia, as laser-ablated
or mechanically wounded necrotic cells are extruded identically
to apoptotic cells (Tamada et al., 2007). Therefore, we added
necrotic cell fragments, prepared by scraping and needle shear-
ing cells and found that they induced accumulation of actin in the
contacted monolayer and with the same kinetics (Fig. 1). When
added to a cell monolayer, 60% of added control cell frag-
ments (green) resulted in actin (red) accumulation in the con-
tacted cells (Fig. 1, A and C). Pre-digestion of the dead cell
fragments with trypsin did not signicantly alter actin accumu-
lation (Fig. 1 C), suggesting that the signal triggering the re-
sponse is not a protein. We then postulated that the signal is a
bioactive lipid.
Based on the fact that a bioactive lipid within this cell
fragment should activate Rho-dependent actomyosin assembly
and contraction during extrusion, we investigated several candi-
date lipids and found that addition of S1P caused actin accumu-
lation when added to monolayers, as in our cell addition assay
(Fig. S1). Conversely, a total lipid extraction from Escherichia
coli that did not contain S1P did not elicit the same reaction.
Based on this experiment, the fact that a precursor to S1P, cer-
amide, is pro-apoptotic (Kolesnick and Hannun, 1999; Hannun
and Obeid, 2002), and recent ndings that S1P is produced in
apoptotic cells (Kolesnick and Hannun, 1999; Hannun and Obeid,
2002; Gude et al., 2008; Weigert et al., 2010), we investigated
whether this lipid is required for extrusion. To directly test if
S1P is required for the actin reaction in the cell addition assay,
we used SKI II, a specic, potent noncompetitive inhibitor of
SphK that blocks conversion of sphingosine to S1P (French
et al., 2003). Pre-treatment of cells with SKI II before making
cell fragments signicantly inhibited the formation of actin
cables when added to monolayers (Fig. 1, B and C). SKI V,
another SphK inhibitor, also reduced the percentage of cell frag-
ments that induce actin accumulations with even stronger ef-
ciency than SKI II (Fig. 1 C and Fig. S2, A and B). Therefore,
we conclude that SphK is required in dying cells to trigger actin
accumulation in the live cells they contact.
To test if SphK is required for extrusion of apoptotic cells
from an MDCK monolayer, we pretreated the monolayer with
SKI II and induced apoptosis with short-wave UV light. Extru-
sion was evaluated by immunostaining the resulting mono-
layers for active caspase-3 Ab to identify apoptotic cells, Alexa
Fluor 568–phalloidin to analyze actin-based extruding rings,
and Hoechst for DNA. Fig. 1 D shows a control extruding apop-
totic cell, focusing only on the dying cell for the caspase-3 and
DNA (which is fragmented) and in another plane on the actin
ring, which is around and beneath the dying cell. Treatment
with SKI II inhibited formation of the actin extrusion ring and
resulted in holes in the monolayer wherever there were apop-
totic cells (Fig. 1 E). Note that when a cell extrudes, DNA and
caspase-3 do not exist in the same plane as its neighboring cells,
whereas all nuclei are in focus when extrusion is blocked. SKI II,
SKI V, and ,-threo-dihydrosphingosine (tDHS), a competitive
Extrusion requires sphingosine-1-phosphate receptor 2 • Gu et al.
Treating WT 3 d post-fertilization (dpf) zebrash with apoptotic
stimuli resulted in extrusion of dying cells (Fig. 3, F and H),
whereas induction of apoptosis in miles apart (mil) zebrash,
which carry a loss-of-function mutation in S1P
(Kupperman et al.,
2000), resulted in apoptotic cells that do not extrude (Fig. 3, G and I,
and Videos 1 and 2). Instead, brightly staining active capase-3
positive cells remain in the plane of the epidermis with no obvious
actin ring around or below them. Of 60 apoptotic cells from WT
zebrash, 59 extruded and one did not, whereas of 88 apoptotic
within the monolayer blocked extrusion of dying cells (Fig. 3 D).
These results support the conclusion that extrusion requires
in the cells surrounding a dying cell, but not in the apop-
totic cell itself.
Our previous work showed that the zebrash larval epider-
mis provides an excellent in vivo model system to study extrusion
(Slattum et al., 2009). To test if S1P
-mediated signaling is also
required for apoptotic cell extrusion in vivo, we tested if zebra-
sh that have a mutation in S1P
could extrude epidermal cells.
Figure 1. Inhibitors of SphKs block actin assembly and apoptotic cell extrusion. (A and B) Alexa Fluor 488–labeled cell fragments (green) prepared from
MDCK cells pretreated with DMSO (A) or SKI II (B) were added to an intact MDCK monolayer. Arrows point to added cell fragments. (C) The percentage of
cell fragments causing actin assembly from three independent experiments; n = 100 cell fragments per experiment and error bars are standard deviations
(SDs). *, P < 0.05; **, P < 0.01. (D and E) Extrusion in an MDCK monolayer in the presence of DMSO (D) or SKI II (E). Arrows point to active caspase-3–
positive dying cells in each case. (F) Quantification of nonextruded active caspase-3–positive apoptotic cells with DMSO or SphK inhibitor treatment from
three independent experiments; n = 100, error bars = SDs. ***, P < 0.001. Bars, 10 µm.
JCB • VOLUME 193 • NUMBER 4 • 2011 670
S1P localizes to the extruding apoptotic
cell and its surrounding epithelial cells
To visualize S1P formation during apoptotic cell extrusion, we
stained UV-irradiated HBE monolayers with anti-S1P mAb and
imaged them by confocal microscopy. Fig. 4 shows representa-
tive 3D projections of images of early, middle, and late stages of
cells from mil zebrash, none extruded. Addition of JTE-013 to
WT zebrash also blocked extrusion of apoptotic epidermal cells
(Fig. S3 C). Of 20 apoptotic cells from WT zebrash treated with
JTE-013, one extruded and the rest did not. These data indicate
that S1P signaling through the S1P
is required for extrusion in a
number of vertebrate epithelia both in vivo and in culture.
Figure 2. An inhibitory anti-S1P mAb blocks apoptotic cell extrusion. (A and B) Extrusion in an MDCK monolayer treated with short-wave UV to induce
apoptosis in the presence of a mouse IgG isotype control (A) or 10 µg/ml anti-S1P mAb (B). (C) Quantification of nonextruded apoptotic cells from three
independent experiments; n = 100 active caspase-3–positive cells where error bars are SDs; **, P < 0.01. (D and E) Alexa Fluor 488–labeled cell frag-
ments (green) prepared from MDCK cells were added to an intact MDCK monolayer in the presence of a mouse IgG isotype control (D) or 10 µg/ml anti-
S1P mAb (E). (F) The percentage of cell fragments causing actin assembly from three independent experiments; n = 100 cell fragments per experiment and
error bars = SDs. ***, P < 0.001. Bars, 10 µm.
671Extrusion requires sphingosine-1-phosphate receptor 2 • Gu et al.
Figure 3. Apoptotic cell extrusion requires the S1P
receptor. (A) HBE cells induced to undergo apoptosis with UV in the presence of DMSO or the in-
dicated S1P receptor antagonists. (B) qRT-PCR confirms shRNA-mediated knockdown of S1P
in HBE cells. (C) Quantification of nonextruded apoptotic
cells in HBE monolayers expressing control or S1P
-specific shRNA after UV treatment. (D and E) A dying HBE cell is not extruded by S1P
-silenced cells
(D, green), but is extruded successfully by normal surrounding cells (E). When S1P
shRNA is only in the dying cell (E), it extrudes and is in a higher plane
than the actin ring below it, but is in the same plane when the surrounding cells are knocked down for S1P
(D). Projections of extruding and nonextruding
apoptotic cells from WT (F) or mil (G) zebrafish larvae, respectively. (H and I) Cross sections (XZs) of an apoptotic extruding cell (H) and a nonextruding
cell (I) from WT (H) and mil zebrafish larvae (I), respectively. For all bar graphs, each bar represents the average percentage of nonextruded apoptotic
cells to total apoptotic cells with each treatment from three independent experiments; n = 100 dying cells per experiment, error bars = SDs. **, P < 0.01;
***, P < 0.001. Bars, 10 µm.
JCB • VOLUME 193 • NUMBER 4 • 2011 672
Figure 4. Apoptotic cells produce and transmit S1P during extrusion. (A–C) Confocal fluorescence images during early (A), middle (B), and late (C) stages
of extrusion of apoptotic cells from an HBE monolayer. (D and E) Confocal fluorescence images of blocked apoptotic cell extrusion by SKI II (D) or the
antagonist JTE-013 (E). Each experimental sample was visualized with five (B and C) or three (A, D, and E) consecutive 3D projections (comprising
2-µm thickness each), as necessary to span the full distance from the most basal to the most apical section (second-to-bottom and top images, respectively).
Note that total cell height under the different conditions varies: during early extrusion (A) and when extrusion is blocked with SKI II and JTE-013 (D and E),
the dying cell is not squeezed out of the epithelium and therefore does not inhabit as great an apical-to-basal distance as when the dying cell is extruding
(B and C). A, B, C, and E were obtained using a confocal microscope (TCS SP5; Leica), whereas D was taken using an inverted microscope (Eclipse
TE300; Nikon) converted for spinning disc confocal microscopy. A–E represent zoomed-in region (square) from each montage. (D) Inset denoting that
the unextruded cell in D is apoptotic. Bars, 10 µm.
673Extrusion requires sphingosine-1-phosphate receptor 2 • Gu et al.
out of the epithelium, thereby detaching it from the epithelium
and underlying matrix. As S1P is known to promote survival
(Kolesnick and Hannun, 1999; Hannun and Obeid, 2002; Weigert
et al., 2010), loss of S1P in the extruded cell may help promote
its death, whereas the increase of S1P in the surrounding cells
could promote their survival, ensuring that they can extrude the
dying cell. How S1P is produced and exported outside the cell
will be important future goals.
Extrusion is critical for maintaining proper epithelial bar-
rier function; therefore, we expect defects in the S1P
pathway may result in diseases associated with poor mucosal
barriers. Aberrant levels of S1P have been associated with asthma,
which many believe may initiate from poor barrier function in
airway epithelia (Gitter et al., 2001; Proksch et al., 2006; Sartor,
2006; Kim et al., 2009; Swindle et al., 2009; Voelkel and Spiegel,
2009). Further, aberrant S1P signaling is known to be involved
in a variety of pathways that could further aggravate asthma, in-
cluding immune cell migration (Matloubian et al., 2004; Gude
et al., 2008; Weigert et al., 2009), epithelial proliferation (Shida
et al., 2008), and vasoconstriction (Watterson et al., 2005). S1P
knockout in mice also results in defective epithelial barrier
function in the cochlea (Kono et al., 2007). Therefore, the iden-
tication of the S1P
pathway in controlling extrusion will
allow us to test if defective extrusion could bring about a variety
of epithelial barrier diseases in animal models.
On the other hand, extrusion could be activated normally
during development or inappropriately during cancer progres-
sion. Several reports have shown that cells can extrude without
dying (Gibson and Perrimon 2005; Shen and Dahmann 2005;
Monks et al., 2008). Further, neuroblasts delaminate from a neuro-
epithelium in Drosophila embryos by a process that appears
similar to basal extrusion (Hartenstein et al., 1994). In advanced
tumors where cell death is blocked or survival signaling is up-
regulated (LoPiccolo et al., 2008; Liu et al., 2009), extrusion
could enable exit of tumor cells to initiate metastasis. Given that
SphK1 is considerably increased in multiple types of cancers
(French et al., 2003; Johnson et al., 2005; Van Brocklyn et al.,
2005) and that S1P is associated with proliferation, survival,
extrusion, where apoptotic cells are marked by condensed and
increasingly fragmented nuclei over time while the actin ring
that surrounds the dying cell gradually constricts inward over
time. Apoptotic cells from early to late stages of extrusion con-
tained prominent intracellular pools of S1P that existed in gran-
ular puncta. No staining was observed in secondary Ab–only
treated cells (not depicted). From early to middle stages of ex-
trusion, S1P was distributed along the plasma membrane pre-
dominantly near the actin ring at the basolateral surface (Fig. 4,
A and B and insets; and Videos 3 and 4). Notably, S1P puncta
could be seen in the surrounding cells, especially during the
middle and late stages of extrusion (Fig. 4 C and Video 5). In-
terestingly, 10% of cells extrude basally instead of apically,
which produce much less S1P than apically extruding cells (not
depicted). As expected, addition of SKI blocked S1P accumula-
tion in and around dying cells, in addition to blocking extrusion
(Fig. 4 D and Video 6). We also note that when signaling via the
was blocked with the antagonist JTE-013, high levels of
S1P accumulated in the dying, unextruded cells but not in sur-
rounding cells (Fig. 4 E and Video 7). Thus, early apoptotic cells
appear to produce and transmit S1P, which is taken up in neigh-
boring cells via the S1P
to drive extrusion.
Taken together, our results suggest that S1P produced by
apoptotic cells binds and activates S1P
in neighboring cells to
trigger contraction of an intercellular actin–myosin ring, which
then squeezes the dying cell out of the monolayer (Fig. 5, see
schematic). Pharmacological inhibition, mutation, or shRNA
silencing of the S1P
receptor signicantly increases the fre-
quency of nonextruded apoptotic cells, suggesting that S1P sig-
nals via S1P
to drive apoptotic cell extrusion.
Based on our data, we propose a model showing how S1P
triggers apoptotic cell extrusion (Fig. 5). S1P produced within
the apoptotic cells is transported to the extracellular surface,
where it binds S1P
on the surface of the surrounding cells and
activates Rho, presumably through p115 RhoGEF (Rosenblatt
et al., 2001; Slattum et al., 2009), to trigger assembly and con-
traction of the actin–myosin extrusion ring at the live–dying cell
interface. Contraction of the extrusion ring drives the dying cell
Figure 5. Model for how S1P triggers extru-
sion. S1P is produced in the dying cell and ex-
ported to neighboring cells. S1P binds to S1P
and triggers formation and contraction of an
actin-and-myosin II extruding ring to squeeze
the dying cell out.
JCB • VOLUME 193 • NUMBER 4 • 2011 674
Apoptotic cells that possessed clearly shrunken nuclei but were not sur-
rounded by a distinguishable actin ring were defined as nonextruded apop-
totic cells. Apoptotic cells that came out of the plane of the monolayer with
strong actin staining around and/or underneath the cells were defined as ex-
truded cells. Old apoptotic cells with strong caspase-3 staining floating above
the monolayer that died before we treated monolayers were excluded.
Fluorescence micrographs of fixed, cultured cells were obtained using a
microscope (DM 6000B; Leica) with an HCX PL Fluotar 63x/1.25 oil lens
(Leica) and captured using a Micromax charge-coupled device camera
(Roper Scientific). IP Laboratory software was used to control the camera
and to process images. Fluorescence micrographs of zebrafish larvae
were obtained using a microscope (model 90i; Nikon) with a 40x PHI lens
(Nikon) and captured using a charge-coupled device camera (Retiga 2000R;
Q Imaging). Confocal micrographs were obtained using a microscope
(TCS SP5; Leica) with a 63x oil lens or an inverted microscope (Eclipse
TE300; Nikon) converted for spinning disc confocal microscopy (Andor
Technologies) using a 60x Plan Fluor 0.95 oil lens with an electron-multiplied
cooled CCD camera 1,000 x 1,000, 8 x 8 mm
driven by the IQ software
(Andor Technologies). We used ImageJ to stack confocal sections into
Z series that were then color combined and reconstructed into 3D image
using MetaMsorph (GE Healthcare). For HBE cells stained with anti-S1P,
we displayed five consecutive projections of 2-µm thickness each using the
“montage” function on MetaMorph software. All images were processed
further using ImageJ, Photoshop (Adobe), Illustrator (Adobe), and Quick-
time Pro (Quicktime) software.
Total RNA was isolated from cultured cells using the RNeasy kit (QIAGEN)
and reverse-transcribed using the SuperScript III first strand synthesis kit
(Invitrogen) with random hexamers according to the manufacturer’s guide-
lines. PCR detection of S1P
was conducted as described previously
(Estrada et al., 2008). In brief, PCR amplification of the targeted fragments
was performed with 30 cycles of denaturation at 95°C (30 s), annealing
at 58°C (30 s), and extension at 72°C (30 s). PCR primer pairs used were:
: sense, 5-GCACCAACCCCATCATTTAC-3, antisense, 5-TTGTCCC-
: sense, 5-TCAG-
: sense, 5-AGCCTTCTGCCCCTCTACTC-3, antisense,
: sense, 5-ACAACTACACC-
tative real-time PCR was performed using a LightCycler480 (Roche) and
the SYBR Green PCR master mix (SABiosciences). Relative mRNA expres-
sion was quantified using the comparative threshold method with the con-
tent of actin mRNA as internal control.
shRNA-mediated gene silencing of S1P
We designed sense and antisense hairpin oligonucleotides specific for S1P
-1) according to a published work (Estrada et al., 2008). Two ad-
ditional pairs of hairpin oligonucleotides were used to knockdown the S1P
receptor. The oligonucleotide sequences were: shS1P
-2: sense, 5-GCGC-
-3: sense, 5-GCAAGTTCCACTCGGCAAT-3, antisense: 5-ATTGC-
CGAGTGGAACTTGC-3. The sense and antisense oligonucleotides were
annealed and cloned into a pLentilox 5.0–based lentiviral vector. Trans-
ducing lentiviral particles carrying the shRNA oligonucleotides were pro-
duced by packaging in 293T cells. Lentiviral particles carrying nonspecific
shRNA oligonucleotides were used as a control. HBE cells were transduced
with lentiviral particles for 6 h in the presence of 2 µg/ml polybrene,
washed, and replaced with fresh medium. Stably transduced cells were
FAC-sorted based on GFP fluorescence.
The statistical analysis was performed using an unpaired t test. Values of
P < 0.05 were considered significant.
Online supplemental material
Fig. S1 shows that S1P directly induces actin assembly at the apex of an
MDCK monolayer. Fig. S2 shows that inhibition of actin assembly and
apoptotic cell extrusion by inhibitors of SphKs is dose dependent. Fig. S3
shows that apoptotic cell extrusion requires the signaling mediated by
. Video 1 shows a tilting 3D projection movie of two apoptotic cells
that do not extrude from the epidermis of a mil zebrafish embryo. Video 2
shows the Z planes of an apoptotic cell that extrudes normally from the
migration, and invasion of cancer cells (Maceyka et al., 2002;
Spiegel and Milstien, 2003; Radeff-Huang et al., 2004), in-
creased S1P signaling could enable tumors to invade by misreg-
ulating extrusion. Therefore, our work dening the signaling
that drives extrusion of an epithelial cell from a layer may also
be important for driving other delamination events crucial to
developmental differentiation or invasion of tumor cells.
Materials and methods
Cell culture
MDCK II cells were cultured in Dulbecco’s minimum essential medium
(DMEM) high glucose with 5% FBS and 100 µg/ml penicillin/streptomycin
(all from Invitrogen) at 5% CO
, 37°C. HBE cells were cultured in MEM
supplemented with 10% FBS and l-glutamine in a flask coated with human
fibronectin type I (BD), bovine collagen I (Purecol; Inamed biomaterials),
and BSA (Invitrogen).
Drug and UV treatment
Cells were treated with 30 µM SKI II (EMD), 4 µM SKI V (Sigma-Aldrich),
40 µM tDHS (Avanti Polar Lipids, Inc.), 10 µM JTE-013 (Tocris Bioscience),
10 µM VPC-23019 (Avanti Polar Lipids, Inc.), 10 µM FTY720-P (Echelon
Biosciences), or 10 µg/ml murine anti-S1P mAb (LPath) for 10 min before
UV treatment. To induce apoptosis, cultured monolayers were exposed to
1,200 µJ/cm
UV254 irradiation in a UV series II (Spectroline) and incu-
bated for 2 h before fixation.
Cell staining
Cells were fixed with 4% formaldehyde in PBS at 37°C for 20 min, perme-
abilized for 10 min with 0.5% Triton in PBS, blocked with AbDil (PBS with
0.1% Triton X-100 and 2% BSA) for 10 min, and incubated with primary
antibody for 1 h. Antibody concentrations used for immunostaining were:
1:200 rabbit anti-active caspase-3 (BD) and 50 µg/ml anti-S1P mAb (LPath
Inc.). Alexa Fluor 488 goat anti–rabbit IgG and Alexa Fluor 488 goat
anti–mouse IgG were used as secondary antibodies to detect active
caspase-3 and S1P, respectively. Actin was detected with Alexa Fluor 568–
phalloidin (Invitrogen). DNA was detected with 1 µg/ml Hoechst 33342
(Sigma-Aldrich) or 5 µM DRAQ5 (Axxora).
Zebrafish staining
To induce apoptosis, we treated 3-d-old zebrafish larvae with 1% DMSO
on ice for 30 min, followed by recovery at 28°C for 10 min. Embryos were
then fixed in 4% formaldehyde, 0.15% glutaraldehyde, 1 mM MgCl
, 0.2%
Triton X-100, and 25 mM Pipes, pH 6.9, for 2 h, blocked in 0.25% casein
overnight. Cells undergoing apoptosis were identified using an anti-activated
caspase-3 rabbit polyclonal Ab (BD) followed by incubation with Alexa Fluor
488 anti–rabbit IgG Ab (Invitrogen). Actin was visualized using 0.1 µg/ml
Alexa Fluor 568–phalloidin (Invitrogen). DNA was visualized using 1 µg/ml
DAPI (Sigma-Aldrich) or 5 µM DRAQ5 (Enzo Life Sciences).
Addition of dead cell fragments or S1P to monolayers
To make dead cell fragments, MDCK cells were scraped from the culture
dish and sheared with a 27G1/2 needle seven times. Cell fragments were
transferred to a microfuge tube and centrifuged for 1 min at 8,000 rpm in a
microfuge (Eppendorf). Cells were fluorescently labeled by resuspending
them in 50 µl DMEM containing 40 µg/ml FITC snail Lectin (Invitrogen) for
5 min and washed three times with 1 ml of DMEM. The labeled cell frag-
ments were then added to confluent MDCK monolayers cultured on glass
coverslips, and incubated for 90 min. The coverslips were fixed and stained
with Alexa Fluor 568–phalloidin and Hoechst dye. For drug inhibition in
the dead cell fragment addition, cells were pretreated for 10 min with SKI
at concentrations listed above before scraping and needle shearing. For
S1P mAb addition, cell fragments were added to MDCK monolayers in the
presence of 10 µg/ml S1P mAb. For the lipid addition experiment, 20 µg/ml
S1P (Avanti Polar Lipids, Inc.) or total E. coli lipid extraction (Avanti Polar
Lipids, Inc.) resuspended in DMEM was added to confluent MDCK mono-
layers, incubated for 1 h, and fixed and stained with phalloidin and
Hoechst dye, as above.
Quantification of cell extrusion
To quantify the ratio of nonextruded apoptotic cells, we counted 100 active
caspase-3–positive cells that were associated with cultured monolayers.
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to extrude from the epidermis of a mil zebrafish embryo. Videos 3–5
show the localization of S1P during early, middle, and late stages of
apoptotic cell extrusion. Video 6 shows that an apoptotic cell does not
produce S1P or extrude in the presence of SKI II. Video 7 shows that
an apoptotic HBE cell that generates high levels of S1P fails to extrude
in the presence of JTE-013. Online supplemental material is available at
We thank Dr. Jean Marie Delalande and Amy Carr for help on zebrafish ex-
periments and to Mark Metzstein, Katie Ullman, and Thomas Marshall for
helpful comments on our manuscript. We also thank Dr. Diana Stafforini for
helpful advice regarding lipids, Dr. Carl Thummel for use of his LSM confocal
microscope, and Dr. James Bear for providing the lentiviral constructs used for
shRNA experiments.
This work was supported by a National Institutes of Health Innovator
Award no. DP2 OD002056-01 to J. Rosenblatt and P30 CA042014
awarded to The Huntsman Cancer Institute for core facilities. R. Sabbadini has
stock options in Lpath, Inc.
Submitted: 14 October 2010
Accepted: 15 April 2011
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    • "Finally, S1P 2 -mediated activation of the Hippo pathway might be pro-cancerous (Miller et al., 2012; Yu et al., 2012; see above). As metastasis of epithelium-derived tumors has been linked to basal rather than apical epithelial extrusion, it is interesting to note that the S1P 2 antagonist, JTE- 013, preferentially inhibited apical extrusion (Gu et al., 2011), although the proposed role of reduced S1P production in basal extrusion of oncogenic K-Ras-expressing cells remains unclear as S1P production by these cells has not been quantified (Slattum et al., 2014). Taken together, apart from the specific situation with S1P 2 in B-cell lymphoma, the presently available studies suggest that the S1P 2 receptor controls mechanisms which both promote and prevent tumor growth, invasion, and metastasis. "
    [Show abstract] [Hide abstract] ABSTRACT: The sphingosine-1-phosphate (S1P) signalling system with its specific G-protein-coupled S1P receptors, the enzymes of S1P metabolism and the S1P transporters, offers a multitude of promising targets for drug development. Until today, drug development in this area has nearly exclusively focused on (functional) antagonists at the S1P1 receptor, which cause a unique phenotype of immunomodulation. Accordingly, the first-in class S1P1 receptor modulator, fingolimod, has been approved for the treatment of relapsing-remitting multiple sclerosis, and novel S1P1 receptor (functional) antagonists are being developed for autoimmune and inflammatory diseases such as psoriasis, inflammatory bowel disease, lupus erythematodes, or polymyositis. Besides the S1P1 receptor, also S1P2 and S1P3 are widely expressed and regulate many diverse functions throughout the body. The S1P2 receptor, in particular, often exerts cellular functions which are opposed to the functions of the S1P1 receptor. As a consequence, antagonists at the S1P2 receptor have the potential to be useful in a contrasting context and different areas of indication compared to S1P1 antagonists. The present review will focus on the therapeutic potential of S1P2 receptor antagonists and discuss their opportunities as well as their potential risks. Open questions and areas which require further investigations will be emphasized in particular.
    Full-text · Article · Jun 2016
    • "S1P expressed by apoptotic cells or RasV12-transformed cells has been reported to be an important regulator for the elimination of those cells from the epithelium (Gu et al., 2011; Slattum et al., 2014). SphKI2 is a specific inhibitor for sphingosine kinase 1 (SphK1) that catalyzes the production of S1P from sphingosine. "
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    • "Retroviral production and infections were as described (Gu et al., 2011). Infected HPAF II or HBE cells 382 were sorted for GFP by using a BD FACSAria Cell Sorter. "
    [Show abstract] [Hide abstract] ABSTRACT: When epithelia become too crowded, some cells are extruded that later die. To extrude, a cell produces the lipid, Sphingosine 1-Phosphate (S1P), which activates S1P2 receptors in neighboring cells that seamlessly squeeze the cell out of the epithelium. Here, we find that extrusion defects can contribute to carcinogenesis and tumor progression. Tumors or epithelia lacking S1P2 cannot extrude cells apically and instead form apoptotic-resistant masses, possess poor barrier function, and shift extrusion basally beneath the epithelium, providing a potential mechanism for cell invasion. Exogenous S1P2 expression is sufficient to rescue apical extrusion, cell death, and reduce orthotopic pancreatic tumors and their metastases. Focal Adhesion Kinase (FAK) inhibitor can bypass extrusion defects and could, therefore, target pancreatic, lung, and colon tumors that lack S1P2 without affecting wild-type tissue. DOI: http://dx.doi.org/10.7554/eLife.04069.001
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