Semaphorin3A signaling controls Fas (CD95)-mediated apoptosis by promoting Fas translocation into lipid rafts

doi:10.1182/blood-2007-06-096529Prepublished online December 3, 2007;   

 Luca Tamagnone and Alfonso CatalanoSimona Moretti, Antonio Procopio, Raffaella Lazzarini, Maria Rita Rippo, Roberto Testa, Maurizio Marra, promoting Fas translocation into lipid raftsSemaphorin3A signaling controls Fas (CD95)-mediated apoptosis by

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Word Count: Abstract 156 Text 4409

Semaphorin3A signaling controls Fas (CD95)-mediated apoptosis by promoting Fas

translocation into lipid rafts

Simona Moretti1,2, Antonio Procopio1,2, Raffaella Lazzarini1,2, Maria Rita Rippo1,2, Roberto Testa3,

Maurizio Marra3, Luca Tamagnone4 and Alfonso Catalano1,2

1Department of Molecular Pathology and Innovative Therapies, Polytechnic University of Marche,

Ancona, Italy

2Center of Cytology and 3Diabetology Unit, Research Department, Italian National Research Centers

on Aging (INRCA-IRCCS), Ancona, Italy

4Institute for Cancer Research and Treatment, University of Turin, Candiolo, Italy

Short Title: Sema3A enhances Fas-mediated apoptosis

Corresponding author: Alfonso Catalano, Dipartimento di Patologia Molecolare, Politecnica delle

Marche, Via Tronto 10/A, 60100, Ancona, Italy. Phone: (39) 0712206245, Fax: (39) 0712206240, E-

mail: [email protected]; [email protected]

Blood First Edition Paper, prepublished online December 3, 2007; DOI 10.1182/blood-2007-06-096529

Copyright © 2007 American Society of Hematology

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Abstract

Semaphorins and their receptors (plexins) have pleiotropic biological functions, including regulation of

immune responses. However, the role of these molecules inside the immune system and the signal

transduction mechanism(s) they utilize are largely unknown. Here, we show that Semaphorin3A

(Sema3A) triggers a proapoptotic programme that sensitizes leukemic T cells to Fas (CD95)-mediated

apoptosis. We found that Sema3A stimulation provoked Fas translocation into lipid raft microdomains

before binding with agonistic antibody or FasL (CD95L). Disruption of lipid rafts reduced sensitivity

to Fas-mediated apoptosis in the presence of Sema3A. Furthermore, we show that plexin-A1, together

with Sema3A-binding neuropilin-1, was rapidly incorporated into membrane rafts after ligand

stimulation, resulting in the transport of actin-linking proteins into Fas-enriched rafts. Cells expressing

a dominant-negative mutant of plexin-A1 did not show Fas clustering and apoptosis upon Sema3A/Fas

co-stimulation. This work identifies a novel biological function of semaphorins and presents an

unexpected signaling mechanism linking semaphorin to the tumor necrosis factor family receptors.

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Introduction

The semaphorin family comprises soluble and membrane-bound proteins that function during neuronal

development, organogenesis, angiogenesis and cancer progression1,2. This family has also attracted the

attention of immunologists as novel regulators of immune cell responses3. Various members of

semaphorins act as amplifiers of the immune response4, while others, the secreted semaphorins of class

3 in particular, may negatively control immune functions5. Previously, we have found that Semaphorin-

3A (Sema3A), a prototype member of secreted semaphorins of class 3, has the potential to inhibit T

cell functions by promoting growth arrest and/or blocking proinflammatory cytokine secretion6. The

downstream pathways by which Sema3A exerts its action on T cells are still unclear, but they are likely

to involve a receptor complex formed from one of two neuropilin (NP) proteins and one of four Plexin-

A proteins. NPs serve as the primary ligand binding sites and Plexin-As as the signal transducing

components7,8.

Recent evidence indicated that Sema3A/NP/PlexA signaling may regulate cell apoptosis9,10. In fact,

upon treatment with recombinant human Sema3A, neurons were found to undergo apoptosis.

Moreover, marked and prolonged protection from dopamine-induced apoptosis was achieved by co-

treatment with function-blocking anti-Sema3A antibodies. Therefore, Sema3A has been proposed to

act as an autocrine and paracrine “amplifier” signal for neuronal cell death. Apoptosis is a pivotal

process in immunological development that is frequently curbed in lymphoid leukemias. Interestingly,

the potential role of Sema3A in the regulation of apoptosis in immune cells has not been investigated

so far. To address this question, we assayed the potential regulatory function of Sema3A in Fas-

induced apoptosis of human leukemic cells.

Fas (CD95/APO-1) is the prototype of “death receptors” in the tumor necrosis factor (TNF) receptor

superfamily. It has been implicated in a wide range of apoptosis-based physiological processes, e.g. T-

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cell-dependent cytotoxicity, deletion of autoreactive T and B cells, activation-induced cell death, tumor

surveillance, immune privilege, angiogenesis, as well as pathological degenerative diseases such as

autoimmunity, fulminant hepatitis, and neurodegeneration11,12. Upon ligation with its cognate ligand

FasL, or with agonistic antibodies, Fas rapidly recruits the Fas-associated death domain protein

(FADD) and procaspase 8, forming the so-called “death inducing signaling complex” (DISC). The

clustering of procaspase-8 molecules in the DISC complex results in their activation by self-cleavage,

triggering downstream effector caspases and eventually leading to apoptosis13. In certain cells

(indicated as “type I”), Fas is localized in detergent-resistant plasma membrane microdomains called

lipid rafts, and Fas-induced apoptosis depends on signaling pathways restricted to these

microdomains14. In contrast, in other cell types (i.e. “type II”), Fas is excluded from lipid rafts and its

signaling depends on the clustering with selected co-receptors15-17. Lipid rafts are cholesterol-rich

structures where adapters and kinases required for signal transduction are localized, including

glycosylphosphatidyl-inositol (GPI)-linked proteins and molecules connecting the actin cytoskeleton to

the plasma membrane (the so-called ERM proteins)18. The formation of membrane platforms where

Fas and its signal-transducers are brought in close proximity is thought to increase DISC formation and

therefore potentiate Fas signaling15-17. Here, we show a novel function of Sema3A, mediated by

plexins, whereby it increases Fas translocation into membrane rafts, sensitizing leukemic cells to Fas-

mediated apoptosis.

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Materials and methods

Cell culture and apoptosis

The human leukemic cell lines HUT78 and HUT78.B119 was kindly provided by R. Testi (University

of Rome, Tor Vergata, Italy). The other cell lines were obtained from American Type Culture

Collection and grown as described previously in RPMI-1640 culture medium supplemented with 10%

heat-inactivated fetal calf serum (FCS).6 Bone marrow aspirates, obtained from patients at the initial

diagnosis and after signing informed consent, were kindly provided by the Clinic of Hematology of the

Polytechnic University of the Marche (Ancona, Italy). Mononuclear cells isolated by Ficoll-Hypaque

density gradient centrifugation, consisting mostly of leukemia blasts (> 90%), were washed in

phosphate-buffered saline (PBS), resuspended in cell culture medium, and used immediately for

experimentation. Death receptor-mediated apoptosis was induced with either rhFasL

(rhsSuperFasLigandTM), TRAIL, anti-Fas mAb APO1-1 (Alexis Biochemicals, Milan, Italy), or

cytotoxic anti-Fas CH11 antibody (Upstate Biotechnology, Milan, Italy). Human recombinant Sema-

3A fused to human Fc fragment (Sema3A-Fc) were purchased from R&D Systems (Minneapolis, MN)

and added simultaneously to anti-Fas stimuli. For the NP1 functional blocking test, the blocking anti-

NP1 antibody, a generous gift of Dr. M. Tessier-Lavigne, was previously described.6 The control

antibody was a rabbit polyclonal anti-NP1 antibody (clone H286, Santa Cruz Biotechnology, Santa

Cruz, CA) with a nonblocking function.6 The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-

fluoromethylketone (zVAD-fmk), the de novo RNA inhibitor actinomycin D or the protein synthesis

inhibitor cycloheximide were from Sigma (Milan, Italy). To disrupt lipid rafts, cells (5 x 105) were

pretreated with 15 µg/ml methyl-β-cyclodextrin (MBCD) (Sigma) for 1 hr at 37°C in serum-free

medium before Sema3A-Fc and Fas stimuli.

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For quantitative determination of apoptosis, cells (5 x 105) were fixed overnight in 70% ethanol at

4°C. Cells were then incubated for 1 hr with 1 mg/mL RNase A and 20 µg/mL propidium iodide at

room temperature, and analyzed with a Becton Dickinson (Milan, Italy) FACScan flow cytometer as

described previously20. Apoptotic cells were calculated as the percentage of cells in the sub-G1 region

in cell cycle analysis. Apoptotic cells were also identified using an annexin-V apoptosis detection kit

(BD-Pharmingen).

Immunofluorescence Flow Cytometry

Cell surface expression of Fas and NP1 was analyzed by immunofluorescence flow cytometry in 4 x

105 cells as described previously20 in a Becton Dickinson FACSCalibur™ flow cytometer using anti-

Fas DX2 mAb (a gift of Dr. R. Testi) and specific antibody against NP1 (Clone H286, Santa Cruz

Biotechnology, Santa Cruz, CA). FAM-VAD-fmk was obtained from Cell Technology, Inc. Isotype-

matched control antibodies were included in each staining.

Isolation of lipid rafts

Lipid rafts were isolated from 107 cells by nonionic detergent lysis and centrifugation on discontinuous

sucrose gradients exactly as described previously15. One ml fractions were collected from the top of the

gradient and 20 µl of each fraction was subjected to SDS-PAGE, immunoblotting, and enhanced

chemiluminescence detection. Location of lipid rafts was determined using cholera toxin (CTx) B

subunit conjugated to horseradish peroxidase (anti-GM1; Sigma) or by blotting fractions for the

tyrosine kinase Lck (obtained from Santa Cruz Biotechnology). Proteins were identified using the

following specific antibodies: anti-48-kDa Fas (C-20), anti-120-kDa-NP-1 (H286), anti-195-kDa-

plexin-A1 (H-60), anti-30-kDa RhoGDI (K-21) rabbit polyclonal antibodies and anti-21-kDa RhoA

mAb (Santa Cruz Biotechnology, Santa Cruz, CA.); anti-29-kDa FADD (clone-1) and anti-80-kDa

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ezrin (clone-18) mAb (BD Transduction Laboratories, Lexington, KY); anti-55-kDa procaspase-8 (Ab-

3) mAb (Oncogene Research Products). Prestained protein molecular mass standards (Bio-Rad; Milan,

Italy) were run in parallel.

Confocal and immunofluorescence microscopy

To confocal microscopy, cells were settled onto poly-L-lysine-coated slides and analyzed with a Zeiss

LSM 510 laser scan confocal microscope (Oberkochen, Germany) for membrane raft and protein

visualization as described15,16. Colocalization assays were analyzed by excitation of the corresponding

fluorochromes in the same section. Negative controls, lacking the primary antibody or using an

irrelevant antibody, showed no staining. To immunofluorescence, cells (3 × 106) were washed and spun

onto glass slides at 900 rpm for 2 min. The cells were then fixed and exposed to purified anti-Fas

(DX2) and anti-plexin-A1 or anti-NP1 antibodies for 1 hr. Then, they were incubated for 30 min with

the secondary antibodies Cy3-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse

IgG (Jackson ImmunoResearch Laboratories, Inc). F-actin filaments were stained with phalloidin-

TRITC and nuclei were stained with DAPI (Sigma, Milan, Italy). Each slide was mounted with

Fluoromount G (EMS, PA). Images were acquired and processed on a fluorescence microscope with

X40 objective. Image analysis and merging of images was done with Adobe PhotoShop 7.0 software

(Adobe System).

Real-Time RT-PCR

Real-Time RT-PCR analysis was done in a Chromo4 sequence detector (Bio-Rad, Milan, Italy) as

previously described6. The primers and probes of Plexin-A1, -A2, -A3 and -A4 genes were determined

by Laboratory Tools software analysis of Stratagene (Milan, Italy). As positive control for human

plexin-A1 and -A4, we used cDNAs derived from PBMC of healthy donors. Details of sequences and

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thermal cycle conditions are available upon request. Data were acquired and analyzed with the

sequence detector Chromo4 software.

Immunoprecipitation and immunoblot analysis

Immunoprecipitation and immunoblots were performed from whole cell lysates as previously

described20-22. Briefly, cells were lysed with EB buffer (20 mM Tris-HCl at pH 7.4, 5 mM EDTA, 150

mM sodium chloride, 10% glycerol and 1% Triton X-100) in the presence of 1 µg/ml leupeptin, 3

µg/ml aprotinin, 1 µg/ml pepstatin, 2 mM phenylmethylsulphonyl fluoride and 1 mM sodium

orthovanadate. After immunoprecipitation with antibodies against NP1 and plexin-A1 (all by Santa

Cruz Biotechnology, Santa Cruz, CA), high-stringency washes were performed (EB buffer containing 1

M lithium chloride). Western blots were then performed and appropriate antibodies were detected

using enhanced chemiluminescence (Amersham Corp., Milan, Italy). In some experiments, blots were

reprobed with an anti-actin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as

loading control.

Dominant-negative plexin-A1 transfectants and RNA interference

Dominant-negative plexin-A1 expression lentiviral vector has been described previously8,21 and was

produced in 293T packaging cells, transiently cotransfected with a mixture of transfer, envelope, and

core-packaging plasmids. Conditioned media containing the vector was harvested 48 h after

transfection, and incubated with a fresh culture of sparse Jurkat cells, in the presence of 8 µg/µl

Polybrene (Sigma, Milan, Italy) for 16 h. The infected cells were previously exposed to 32 µM

genistein for 3 hrs to enhance lentiviral vector entry23. Genistein was purchased from Calbiochem

(Milan, Italy) and diluted in dimethyl sulfoxide (DMSO) prior to use. The final concentration of

DMSO in the cultures never exceeded 0.2% (vol/vol). The plexin construct included a vesicular

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stomatitis virus (VSV) tag, detectable with an anti-VSV-G monoclonal antibody (V-5507; Sigma, St

Louis, MO) by immunoblotting.

Four siRNA sequences specific for human plexin-A1 were selected, synthesized and annealed by the

manufacturer (Dharmacon, Milan, Italy). Transfection was performed using RNAiFect (Qiagen)

according to the manufacturer’s protocol. After 48 h of incubation, the resulting cells were harvested,

washed and used for subsequent experiments. Transfection efficiencies were determined using

fluorescein-labelled non-silencing RNA (35-45%).

Statistical analysis

All values were expressed as mean ± s.e.m of no less than triplicate measurements of three independent

experiments. Comparison of results between different groups was performed by one-way analysis of

variance, paired t-test and ANOVA using StatView 5.0 (NET Engineering, Pavia, Italy). A P value ≤

0.05 was considered statistically significant.

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Results

Pro-apoptotic activity of Sema3A in association with Fas-stimulation

The pro-apoptotic activity of Sema-3A was assessed in a variety of human tumor cells. We first used

Fas-sensitive T leukemia cells Jurkat (as type II cell line) and HUT78 (as type I cell line), as well as the

Fas-resistant HUT78.B1 cells, previously derived from HUT7814. As reported for type I and type II

cells15, Jurkat cells expressed less Fas on their cell surface than HUT78 or HUT78.B1 cells (Figure

1A). rhFasL or the agonistic cytotoxic CH11 anti-Fas antibody induced apoptosis in Jurkat and HUT78

cells, but not in HUT78.B1 cells (Figure 1, B and C). In response to APO1-1, an IgG1 isotype switch

variant of anti-Fas antibody that induces apoptosis in type I but not in type II cells15, HUT78 cells

underwent apoptosis, whereas Jurkat and HUT78.B1 cells were almost completely resistant (Figure 1,

B and C). In contrast, all of the cells were sensitive to TNF-related apoptosis-inducing ligand (TRAIL)

(Figure 1, B and C). When Jurkat (type II) cells were treated with either rhFasL, CH11 or APO1-1, the

co-stimulation with Sema3A strongly increase the sensitivity to cell death (Figure 1B). Sema3A was

less effective in sensitizing HUT78 (type I) cells, whereas it had no effect on HUT78.B1 cells (Figure

1C). Moreover, Sema3A did not increase the sensitivity to cell death induced by TRAIL (Figure 1, B

and C). These data show that Sema3A sensitizes some cell-types to die via anti-Fas stimuli.

We next investigated the involvement of Sema3A-receptor NP1 in the pro-apoptotic activity of

Sema3A. Jurkat, HUT78 and HUT78.B1 expressed similar protein levels of NP1 on their cell surface

(Figure 2A). However, the addition of blocking antibodies directed against NP1 (anti-NP1) abrogated

the sensitizing effect mediated by Sema3A, but had no effect on Fas-induced cell death (Figure 2B).

These data suggest that Sema3A, through NP1 receptor, enhances the death signal induced by Fas

activation, especially in Jurkat (type II) cells.

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Sema3A increases Fas-mediated caspase activation in type II, but not in type I cells

We then analyzed the effect of Sema3A co-treatment on the cleavage of procaspase-8 and -3, which is

the hallmark of Fas-induced apoptosis11-13. In a time-course experiment, we observed full cleavage of

caspase-8 and -3 after only 2 hours of Sema3A/anti-Fas co-stimulation in Jurkat cells (Figure 3A),

while APO1-1 alone was only partly effective at this stage. In contrast, we did not find a substantial

shift in the activation of caspase-8 and -3 in HUT78 cells. In H9 T lymphoma cells, another type I cell

line, there was no change in caspases activation after Sema-3A co-treatment; whereas in the type II

CEM T leukemia cells we found complete cleavage of caspase-8 and -3, similar to Jurkat cells (data

not shown). Consistently, the enhancement of Fas-mediated apoptosis by Sema3A was blocked by pre-

incubating Jurkat or CEM cells with the broad-range caspase inhibitor zVAD-fmk (Figure 3B).

Therefore, the observed differences in Fas-mediated cell death in presence of Sema3A in type II cells

seem to correlate with the intracellular activation of caspases.

Increased Fas-mediated apoptosis by Sema3A does not require new protein synthesis or Rap1

activity

We next examined whether Sema3A could induce Fas up-regulation on the cell surface, resulting in

higher receptor density and signaling potential. However, Sema3A did not increase Fas expression on

the cell surface in Jurkat cells, after up to 6 hours of treatment (at the stage in which the increased

sensitivity to apoptosis was observed; Supplementary Figure 1A). To assess whether the pro-apoptotic

activity of Sema3A was dependent on new protein synthesis, we pretreated Jurkat cells with

actinomycin D (Act. D) or cycloheximide (CHX). Neither Act. D nor CHX were able to inhibit the

increased sensitivity to cell death mediated by Sema3A (Supplementary Figure 1B).

Moreover, since we have previously shown that the small G protein Rap1 is involved in NP1-

mediated Sema3A inhibitory signaling in T-cells6, we asked whether the same signal transducer could

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be implicated in the regulation of Fas-induced apoptosis. We expressed a dominant-negative Rap1

(Rap1N17) in Jurkat cells; however Sema3A co-treatment promoted apoptosis in cells expressing

Rap1N17 in a comparable manner as in cells transfected with an empty vector (Supplementary Figure

1C). Therefore, the pro-apoptotic signaling of Sema3A occurred independently of Rap1 activation.

Sema3A stimulation recruits Fas into the lipid rafts, where semaphorin receptor NP1 is localized

In the absence of the cognate ligand, redistribution of Fas on lipid rafts is a potential mechanism to up-

regulate its signaling in type II cells15-17. Therefore, we asked whether Sema3A may enhance Fas-

mediated apoptosis in Jurkat (type II) cells by Fas redistribution in lipid rafts. The membrane proteins

from Jurkat cell lysates were separated by sucrose gradient ultra-centrifugation and analyzed by

western blotting as previously described15. Fractions containing the lipid rafts (4-5) were identified by

the presence of the tyrosine kinase Lck (Figure 4A). In un-stimulated cells, Fas was not found in lipid

rafts, whereas after 60 min treatment with Sema3A, a marked proportion of it translocated into lipid

rafts (Figure 4A). Neither FADD nor procaspase-8 were recruited into lipid rafts upon Sema3A

stimulation (Figure 4A), consistent with the notion that Fas re-localization into lipid rafts in type II

cells occurs before any stimulation with death receptor ligands15,16. The Sema3A receptor NP1 was

markedly present in lipid rafts before Sema3A treatment and its localization did not change upon

stimulation (Figure 4A). Also in this case, Fas translocation from soluble (S, fraction 11) to insoluble

lipid raft (R, fraction 4) was NP1 dependent, because it was entirely abrogated in the presence of anti-

NP1 blocking antibody (Figure 4B). The raft-associated glycosphingolipid GM1 showed localization of

the raft fraction in these experiments (Figure 4B). Therefore, a portion of NP1 is distributed into lipid

rafts, and its stimulation with Sema3A elicits the recruitment of Fas into lipid rafts.

As previously described16, the disruption of lipid rafts by cholesterol depletion using methyl-β-

cyclodextrin (MBCD) did not affect Fas-mediated apoptosis in Jurkat cells (Figure 4C). However, by

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pre-treating with MBCD we completely abrogated Sema3A-mediated amplification of Fas apoptosis

(Figure 4C). These results indicated that Fas translocation into lipid rafts induced by a NP1-dependent

Sema3A signaling is required to increase Fas-mediated apoptosis.

Sema3A co-stimulation enhances Fas-induced apoptosis in primary leukemic cells

Our data indicated that Sema3A can sensitize some leukemic cell lines to Fas-mediated apoptosis. To

extend our findings to primary leukemic cells, we studied bone marrow cells derived from patients with

acute myeloblastic leukemia or acute promyelocytic leukemia (M2 or M3, respectively, following the

FAB classification). These M2 and M3 leukemia cells were positive for Fas and NP1 (>60% and >50%

positive cells, respectively; data not shown), and underwent apoptosis after Fas stimulation

(Supplementary Figure 2A). Consistent with that seen in Jurkat cells, primary leukemia cells were

sensitized to Fas-induced cell death by treatment with Sema3A, and this effect was inhibited by MBCD

(Supplementary Figure 2A). Staining of leukemia cells with the fluorescent caspase substrate FAM-

VAD-fmk showed that a higher percentage of cells receiving anti-Fas and Sema3A co-treatment

contained more activated caspases than cells receiving anti-Fas alone. Consistently, this effect was

blocked by MBCD pretreatment (Supplementary Figure 2B). Therefore, Sema3A and Fas stimuli may

act in synergy to enhance caspase activation and apoptosis in primary leukemic cells.

The Sema3A co-receptor plexin-A is involved in enhancing Fas-mediated cell death

In addition to NP1, members of the plexin-A family (plexin-A1, -A2, -A3, and -A4) serve as

components of receptor complexes for Sema3A.8 We found that plexin-A1 and -A3 mRNAs are

expressed in cell lines HUT78 and H9 (type I), Jurkat and CEM (type II), as well as in primary M2 and

M3 leukemic cells. In contrast, plexin-A2 and -A4 were virtually absent in these cells (Figure 5A).

Plexin-A1 was expressed more abundantly than plexin-A3, and it was confirmed at the protein level by

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immunoblotting (Figure 5B). Moreover, by immunofluorescence microscopy, plexin-A1 and NP1 were

found to co-localize with Fas in Sema3A-treated Jurkat cells, but not in untreated cells (Figure 5, C and

D). A substantial proportion of plexin-A1 translocated into lipid rafts in cells stimulated with Sema3A

(Figure 6A) and the association of plexin-A1 with NP1 was increased in response to Sema3A (Figure

6B). Since we found that NP1 is constitutively present in these membrane microdomains (see Fig. 4),

our data indicate that Sema3A enhances NP1/plexin-A1 complex formation into lipid rafts.

To determine the role of plexin-A1 in Sema3A-mediated signaling, we generated a clone from the

leukemic Jurkat cell line that express a previously characterized plexin-A1 dominant-negative form8,21

(DN-plexin-A1). The control and DN-plexin-A1-transfected cells displayed similar sensitivities to Fas

stimulation alone. However, the DN-plexin-A1 cells was less sensitive to Fas/Sema3A co-triggering

than control cells (Figure 6C). We also selectively knocked down plexin-A1 gene expression. RNAi

against plexin-A1 reduced the pro-apoptotic activity of Sema3A on Jurkat cells (Figure 6D). These

results suggest that NP1 and plexin-A1 are functional receptor components for Sema3A.

Fas re-localization induced by Sema3A requires actin cytoskeleton

We next asked how Fas is concentrated in lipid rafts by Sema3A/NP/PlexA signaling. Clustered rafts

are often bound to the cytoskeleton, and Sema3A signaling is thought to alter actin reorganization in

immune cells24,25. To investigate whether Sema3A could modify the actin cytoskeleton in human

leukemic cells, Jurkat cells treated with Sema3A were stained with phalloidin-TRITC for F-actin and

with Hoechst 33258 to assess nuclear morphology and visualized by fluorescence microscopy. Similar

to FasL, Sema3A caused both cell shape change and a redistribution of the actin cytoskeleton (Figure

7A). In addition, by pre-treating Jurkat cells with the microfilament-disrupting agent cytochalasin-B,

we prevented the sensitization to Fas-induced apoptosis mediated by Sema3A (Figure 7B).

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Ezrin, a major component of the ERM protein family (ezrin, radixin, moesin), can interact with Fas

and mediate Fas cell membrane localization during Fas-induced apoptosis18. We found that ezrin as

well as actin dynamics regulatory proteins, such as RhoA and RhoGDI, were accumulated in lipid rafts

following Sema3A stimulation (Figure 7C). In comparison, the amount of either ezrin, RhoA or

RhoGDI redistributed in the lipid rafts was very faint when the DN-plexin-A1 cell line was stimulated

with Sema3A alone (Figure 7D). In addition, Fas re-localization into lipid rafts was not observed in

these cells upon Sema3A treatment (Figure 7E). All together, these findings indicate that PlexinA-

driven Fas translocation into lipid rafts sensitizes leukemic cells to apoptosis and that this effect seems

to require actin-linking proteins accumulation in membrane rafts and cytoskeleton reorganization.

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Discussion

The recruitment of Fas to lipid rafts, elicited by independent non-apoptotic receptor signaling,

represents a non-completely-understood pathway that modulates Fas-induced cell death14-16.

Semaphorins play a central role in axonal guidance, and emerging evidence points to diverse functions

of several semaphorins (including Sema3A) in the immune system. For instance, class IV semaphorins

(e.g. Sema4D and Sema4A) play crucial roles in the reciprocal stimulation between T cells and APCs,

both in vitro and in vivo26. In addition, Sema3A inhibits the migration of human monocytes in response

to cytokine stimulation27 as well as T cell proliferation and cytokines production under stimulating

conditions6. In this study, we identify a novel biological function for semaphorins in immune cells and

an unexpected signaling mechanism, namely, the coupling of Sema3A to a death receptor.

A pivotal role of secreted semaphorins in the regulation of neuronal cell death has been previously

established28, although the implicated pathways have not been characterized as yet. The present study

demonstrates that secreted Sema3A and its receptor NP1 are important determinants of leukemic cells

sensitivity to Fas-mediated death signals (Figs. 1 and 2). De novo protein synthesis is not essential for

the pro-apoptotic activity of Sema3A, but the redistribution and clustering of membrane-bound Fas into

lipid raft microdomains is a pivotal step for cell death signaling (Fig. 4).

Membrane rafts could serve to generate high local concentration of Fas, as platforms for coupling

adaptor and effector proteins required for Fas downstream signaling, facilitating and amplifying

signaling processes by transient local assembly of various cross-interacting molecules15-17. This is of

particular importance in Fas-mediated signal transduction because death receptors lack enzymatic

activity and the pathway is triggered by protein/protein interactions. The presence of Fas in lipid rafts is

restricted to cells previously described as type I, which show more efficient formation of death

signaling complexes and greater sensitivity to Fas stimuli. In peripheral T cells, it has been shown that

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the membrane distribution of Fas is dynamically regulated by antigen receptor signaling and possibly

by other signals 15-17. This can increase Fas-mediated apoptosis in type II cells and may be important to

promote the clonotypic elimination of chronically stimulated T cells. As some leukemic primary cells

are described as type II cells, in which Fas signaling is dependent on receptor-mediated clustering in

membrane microdomains18, our observations could be also relevant for apoptotic pathways in leukemia

cells. Notably, Sema3A expression is reduced in primary leukemic cells, whereas they express the

receptor NP1 at high levels.29,30 Therefore, the Sema3A-dependent regulatory mechanism described

here may be relevant to curb uncontrolled T-cell proliferation.

The intracellular domain of NP1 is short, and apparently unable to mediate functional responses to

Sema-3A.1 Plexin-A1 is a co-receptor for Sema-3A in the nervous system2-4 and it has a signaling role

in immune responses31,32. We detected plexin-A1 expression in our cells and a dominant negative

mutant of plexin-A1 or siRNA against plexin-A1 blocked the pro-apoptotic activity of Sema3A.

Therefore, Sema-3A may act through plexin-A1 in leukemic cells.

Interestingly, our data show the translocation of plexin-A1 into membrane rafts upon Sema3A

stimulation, while a remarkable fraction of NP1 appears to be constitutively localized in these

membrane microdomains. Since lipid rafts are specialized structures involved in several biological

processes, such as apoptosis, synaptic transmission, adhesion and migration15-18, the clustering of NP1

and Plexin-A1 into these microdomains on the cell surface may lead to a more effective Sema3A

signaling. The clustering of NP1 and plexin-A1 on the cell surface, upon Sema3A stimulation, had

been shown previously in neuronal cells33, however the identity of the implicated membrane

microdomains was not known. Notably, we found that Plexin-A1 is rapidly incorporated into lipid

rafts, after a few minutes of incubation with Sema3A (Fig. 6), and this process precedes Fas clustering

(our unpublished data). Thus, we propose that Sema3A/NP1/Plexin signaling rearranges membrane

rafts, promoting receptor clustering and Fas redistribution.

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How Plexin can affect Fas translocation into lipid rafts is presently unknown. This signal is distinct

from the Rap-1-dependent pathway leading to inhibition of T cell proliferation and cytokine

production6. It has been demonstrated that the cytoplasmic domain of plexins carries an intrinsic R-Ras

GAP activity34, and several groups have reported that plexin-associated effector molecules control

cytoskeletal dynamics and integrin function through monomeric G proteins35. Cytoskeletal

rearrangements or changes in the interaction between Fas and components of the actin cytoskeleton

may contribute to Fas translocation into lipid rafts36. Our findings indicate the involvement of actin-

network remodeling triggered by plexins in the translocation of Fas into lipid rafts (Fig. 7).

Alternatively, post-translational modification of Fas may also mediate translocation, and this point is

currently under investigation.

In conclusion, the data presented here indicate that Sema3A plays a relevant role by bolstering Fas-

mediated apoptosis in human leukemic cells. Moreover, since NP1 and Sema3A are constitutively

expressed in human thymus in both thymic epithelial cells and CD4/CD8-defined thymocytes37, our

data suggest a possible involvement of Sema3A in T cell homeostasis, a process in which the role of

tumor necrosis factor family receptors is well documented11,12.

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Acknowledgments

We acknowledge Dr. M. Tessier-Lavigne (Howard Hughes Medical Institute, Stanford University, CA)

for providing the blocking anti-NP1 antibody. Dr. L. Capparuccia (IRCC, Candiolo, Torino), Dr. G.

Fulgenzi (P.U. of Marche, Ancona, Italy) and Dr. M. Fanelli (Centre of Biotechnology, University of

Urbino, Fano, Italy) for technical assistance. This work has been supported by grants from the Italian

Association for Cancer Research (AIRC) to A.P. and to A.C.; the Ministry of University and the

Ministry of Health to A.P. S.M. and R.L. were supported by a fellowship from AIRC and FIRC,

respectively.

Author contributions

S.M. performed cell-culture experiments, generated figures, and helped to write the manuscript. A.P.

analyzed the data, and helped to design the study and write the manuscript. R.L. assisted with

experiments and provided expertise in molecular biology. R.T., M.M. and M.R.R. provided

methodological expertise and helped to design some experiments. L.T. analyzed the data, and helped to

design the study and write the manuscript. A.C. (principal investigator) designed the experiments,

interpreted and analyzed data, and drafted and edited the manuscript.

The authors declare no competing financial interests.

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Figure legends

Figure 1. Fas-induced cell death varies with cell type in the presence of Sema3A. (A) Jurkat, HUT78

and HUT78.B1 cells were assayed by flow cytometry for their respective cell surface content of Fas. (B

and C) Jurkat, HUT78 or HUT78.B1 cells were stimulated with 50 ng/ml rhFasL, 200 ng/ml CH11,

150 ng/ml APO1-1, and 50 ng/ml TRAIL, in the presence of soluble control IgG or Sema3A-Fc (150

ng/ml) for 7 hrs. Cell death was assessed by staining using FITC-annexin V. Data are the mean ± s.e.m.

of three independent experiments. * indicates P < 0.05 vs. none and ** indicates P < 0.05 vs its

respective death receptor ligand.

Figure 2. The pro-apoptotic activity of Sema3A is dependent by NP1. (A) Surface expression of NP1

for Jurkat, HUT78 or HUT78.B1 cells. (B) Jurkat cells were incubated with CH11 plus Sema3A-Fc or

control IgG for 7 hrs, and cell death was determined as described in figure 1. Alternatively, cells were

first incubated with the blocking anti-NP1 antibody or a control Ab. Statistical analysis is shown.

Figure 3. Sema3A co-signal differentially increases the Fas-mediated caspase activation in cell lines.

(A) Jurkat (left) or HUT78 (right) cells were stimulated with 150 ng/ml APO1-1 in the presence of

control IgG or Sema3A-Fc (150 ng/ml) for indicated times. Activation of caspase 8 and caspase 3 was

analyzed by immunoblotting. The 18 kDa and 17 kDa bands correspond to active cleavage forms of

caspase-8 and -3, respectively. The results shown are representative of three independent experiments.

(B) Jurkat or CEM (type II) cells were incubated for 7 hrs as described in panel A with or without

zVAD-fmk (20 µM). Cell death was assessed by flow cytometry with propidium iodide.

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Figure 4. Sema3A stimulation induces redistribution of Fas into lipid rafts. (A) Jurkat cells (1 x 107)

were treated with control IgG or Sema3A-Fc for 60 min and subjected to density gradient fractionation.

Fractions were immunoblotted with antibodies for Lck, Fas, FADD, caspase-8 and NP1. Fractions

corresponding to lipid raft (R) and soluble (S) proteins are indicated. (B) Jurkat cells were pre-

incubated with the blocking anti-NP1 antibody or a control Ab. Then, cells were treated as in panel A.

Fractions 4 (R) and 11 (S) were immunoblotted with antibodies specific for GM1 and Fas. Data in A

and B are representative of at least three independent experiments. (C) Jurkat cells were stimulated

with Sema3A-Fc for 60 min, depleted of cholesterol with 15 µg/ml MBCD for 10 min at 37 °C and

then stimulated with CH11 for 6 hrs. Cell death was determined by flow cytometry with annexin V

staining. P value is shown.

Figure 5. Plexins expression in leukemic cells. (A) Total RNA (50 ng/µl) was isolated from the

indicated cells and real-time PCR was done with primers and probes specific for plexin-A1, -A2, -A3,

and -A4 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Plexins mRNA expression

was normalized to GAPDH for each sample. (B) Total lysates (50 µg) from indicated cell lines were

immunoblotted (Blot Ab) with anti-plexin-A1 antibody. Expression of actin was used as loading

control. (C and D) Colocalization of Fas (green) with either plexin-A1 (C) or NP1 (D) (red) in

Sema3A-treated Jurkat cells in contrast to cells treated with control IgG. Images are magnified × 1600.

Figure 6. Involvement of Plexin in Sema3A signaling. (A) Jurkat cells were treated with control IgG or

Sema3A-Fc for 60 min, and subjected to density gradient fractionation. For anti-Lck immunoblots, 10

µg of protein was loaded per lane and for anti-plexin-A1 immunoblots, 40 µg of protein was loaded per

lane. (B) Jurkat cells (107 cells per test) were treated with control IgG or Sema3A-Fc (150 ng/ml) for 5

min and lysates were prepared. Equivalent amounts of whole-cell lysates were either

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immunoprecipitated with anti-plexin-A1 antibody (IP: anti-plexin-A1) or anti-NP1 antibody (IP: anti-

NP1). Immune complexes were then immunoblotted (Blot Ab) as indicated. Data in A and B are

representative of three experiments. (C) The Jurkat cell line was engineered by lentiviral-mediated

gene-transfer of a truncated form of plexin-A1, lacking its cytoplasmic domain (DN-plexin-A1). These

cells or control Jurkat cells were incubated with CH11 or CH11 plus Sema3A-Fc for 6 hrs. (D)

Additionally, Jurkat cells transfected with siRNA specific for plexin-A1 or non-silencing siRNA were

cultured with control IgG or Sema3A-Fc for 6-8 hrs. plexin-A1-expression in RNAi-treated Jurkat cells

was determined by RT-PCR analysis. Cell death was quantified by flow cytometry with annexin V

staining. P values were also shown.

Figure 7. Plexin-mediated Sema3A stimulation induces cytoskeleton reorganization and redistribution

of actin-linking proteins into lipid rafts. (A) Jurkat cells were incubated with control IgG or Sema3A-

Fc (150 ng/ml) for 30 min. As positive control, Jurkat cells were also incubated with 50 ng/ml rhFasL.

Actin morphology was visualized by staining with phalloidin-TRITC. Nuclei were stained with

Hoechst 33258. Images are magnified × 1600. (B) Jurkat cells were pretreated with 5 µg/ml

cytochalasin B (CB) for 30 min and then incubated with anti-Fas or anti-Fas plus Sema3A-Fc for 6 hrs.

Cell death was quantified by flow cytometry with annexin V staining. P values were also shown. (C-E)

Control or dominant negative plexin-A1 expressing cells (DN-plexin-A1) were treated with control

IgG or Sema3A-Fc for 60 min and subjected to density gradient fractionation. (C and D) Fractions

were immunoblotted with antibodies for GM1, Ezrin, RhoA, RhoGDI. Western blots were also probed

with anti-VSV-G antibody to detect the VSV-G epitope tag of plexin construct. (E) Fractions derived

from DN-plexin-A1 were immunoblotted with antibodies for Lck and Fas. Lipid raft (R) and soluble

(S) fractions are indicated.

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FIGURE 1

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FIGURE 2

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FIGURE 3

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FIGURE 4-AB

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FIGURE 4C

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FIGURE 5-AB

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FIGURE 5-CD

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FIGURE 6

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FIGURE 7-AB

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FIGURE 7-CDE

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