Importin β1 protein-mediated nuclear localization of death receptor 5 (DR5) limits DR5/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cell death of human tumor cells.

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5)-mediated cell death plays an important role in the elimination of tumor cells and transformed cells. Recently, recombinant TRAIL and agonistic anti-DR5 monoclonal antibodies have been developed and applied to cancer therapy. However, depending on the type of cancer, the sensitivity to TRAIL has been reportedly different, and some tumor cells are resistant to TRAIL-mediated apoptosis. Using confocal microscopy, we found that large amounts of DR5 were localized in the nucleus in HeLa and HepG2 cells. Moreover, these tumor cells were resistant to TRAIL, whereas DU145 cells, which do not have nuclear DR5, were highly sensitive to TRAIL. By means of immunoprecipitation and Western blot analysis, we found that DR5 and importin β1 were physically associated, suggesting that the nuclear DR5 was transported through the nuclear import pathway mediated by importin β1. Two functional nuclear localization signals were identified in DR5, the mutation of which abrogated the nuclear localization of DR5 in HeLa cells. Moreover, the nuclear transport of DR5 was also prevented by the knockdown of importin β1 using siRNA, resulting in the up-regulation of DR5 expression on the cell surface and an increased sensitivity of HeLa and HepG2 cells to TRAIL. Taken together, our findings suggest that the importin β1-mediated nuclear localization of DR5 limits the DR5/TRAIL-induced cell death of human tumor cells and thus can be a novel target to improve cancer therapy with recombinant TRAIL and anti-DR5 antibodies.

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Importin ?1 Protein-mediated Nuclear Localization of Death
Receptor 5 (DR5) Limits DR5/Tumor Necrosis Factor
(TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced
Cell Death of Human Tumor Cells*□
Receivedforpublication,September30,2011 Published,JBCPapersinPress,October21,2011,DOI10.1074/jbc.M111.309377
Yuko Kojima‡1, Masafumi Nakayama§¶, Takashi Nishina§?, Hiroyasu Nakano§**, Makoto Koyanagi§,
Kazuyoshi Takeda§, Ko Okumura§?, and Hideo Yagita§
Fromthe‡LaboratoryofBiomedicalImagingResearch,BiomedicalResearchCenter,§DepartmentofImmunology,?Atopy
ResearchCenter,and**LaboratoryofMolecularandBiochemicalResearch,BiomedicalResearchCenter,JuntendoUniversity
SchoolofMedicine,2-1-1Hongo,Bunkyo-ku,Tokyo113-8421,Japanand¶DepartmentofImmunobiology,Instituteof
Development,AgingandCancer,TohokuUniversity,4-1Seiryo-cho,Aoba-ku,Sendai-shi,Miyagi 980-5875, Japan
S
Background: Nuclear localization of DR5 was observed in TRAIL-resistant tumor cells in human.
Results: TRAIL-resistant tumor cells were sensitized to TRAIL by knockdown of importin ?1.
Conclusion: Importin ?1-mediated nuclear translocation of DR5 limits DR5/TRAIL-induced cell death of human tumor cells.
Significance:ThisprovidesanovelstrategytoimprovetheefficiencyofrecombinantTRAILandanti-DR5antibodiesincancer
therapy.
Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL)/death receptor 5 (DR5)-mediated cell death plays an
important role in the elimination of tumor cells and trans-
formed cells. Recently, recombinant TRAIL and agonistic anti-
DR5monoclonalantibodieshavebeendevelopedandappliedto
cancer therapy. However, depending on the type of cancer, the
sensitivity to TRAIL has been reportedly different, and some
tumor cells are resistant to TRAIL-mediated apoptosis. Using
confocal microscopy, we found that large amounts of DR5 were
localized in the nucleus in HeLa and HepG2 cells. Moreover,
thesetumorcellswereresistanttoTRAIL,whereasDU145cells,
whichdonothavenuclearDR5,werehighlysensitivetoTRAIL.
By means of immunoprecipitation and Western blot analysis,
we found that DR5 and importin ?1 were physically associated,
suggesting that the nuclear DR5 was transported through the
nuclear import pathway mediated by importin ?1. Two func-
tional nuclear localization signals were identified in DR5, the
mutation of which abrogated the nuclear localization of DR5 in
HeLa cells. Moreover, the nuclear transport of DR5 was also
prevented by the knockdown of importin ?1 using siRNA,
resultingintheup-regulationofDR5expressiononthecellsur-
face and an increased sensitivity of HeLa and HepG2 cells to
TRAIL. Taken together, our findings suggest that the importin
?1-mediated nuclear localization of DR5 limits the DR5/
TRAIL-inducedcelldeathofhumantumorcellsandthuscanbe
a novel target to improve cancer therapy with recombinant
TRAIL and anti-DR5 antibodies.
Tumor necrosis factor (TNF)-related apoptosis-inducing
ligand(TRAIL),2amemberoftheTNFfamily,isatypeIItrans-
membraneproteinexpressedonthecellsurfaceofnaturalkiller
cells, T lymphocytes, macrophages, and dendritic cells, and its
mRNA is induced by type I interferons (1–4). Death receptor 5
(DR5), one of the TRAIL receptors and a member of the TNF
receptorsuperfamily,isa48-kDatypeItransmembraneprotein
consisting of 440 amino acids and has a death domain (DD) in
its cytoplasmic region like death receptor 4 (DR4), Fas, and
TNF receptor 1 (1, 5–9). Similar to Fas and DR4, DR5 is gener-
ally expressed in both normal and malignant cells (5). TRAIL
triggers apoptosis through DR4 and/or DR5 in humans or
through DR5 in mice and is an important molecule in tumor
suppression through its ability to induce apoptosis in tumor
cells and transformed cells without obvious damage to normal
cells (1, 4, 6). The TRAIL-induced apoptosis signaling pathway
has been extensively characterized (1, 6, 7). The ligation of
homotrimeric TRAIL to DR5 results in the trimerization of
DR5, the clustering of the DR5 intracellular DD, and the
recruitment of Fas-associated death domain (FADD), leading
to the formation of death-inducing signaling complex (1).
Death-inducing signaling complex moves into a membrane
compartment enriched in lipid rafts and linked to the cytoskel-
eton, therefore initiating signal transduction through intracel-
lular signaling machinery involving the activation of caspases
(1, 2). Procaspase-8 is dimerized by its interaction with Fas-
associated death domain, and it is activated through interchain
cleavage at two cleavage sites. Furthermore, the activated
* This work was supported by Grants-in-aid for Scientific Research 19590257
and22240089fromtheMinistryofEducation,Culture,Sports,Scienceand
Technology, Japan.
□
STheon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental Materials and Methods and Figs. 1 and 2.
1Towhomcorrespondenceshouldbeaddressed.E-mail:kojima-y@juntendo.
ac.jp.
2Theabbreviationsusedare:TRAIL,tumornecrosisfactor-relatedapoptosis-
inducingligand;DR,deathreceptor;DD,deathdomain;FADD,Fas-associ-
ated death domain; NLS, nuclear localization signal; rTRAIL, recombinant
human TRAIL; pAb, polyclonal antibody; PE, phycoerythrin; WGA, wheat
germ agglutinin; WST, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)[2H]tetrazolium monosodium salt; ER, endoplasmic
reticulum.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 50, pp. 43383–43393, December 16, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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caspase-8 cleaves the procaspase-3 dimer at one cleavage site,
and finally, the now active caspase-3 dimer executes apoptosis
(10). In some cell types, when the activation of caspase-8 is not
sufficient, amplification is required and involves the activation
of other caspases. In the current model, the intracellular signal
transduction is regulated by complex mechanisms, and the
resistance to TRAIL-induced cell death can occur at several
points in the signal pathways (1, 11). However, in the course of
inducing cell death in tumor cells, DR5 expression on the
plasma membrane is required because the TRAIL/DR5-medi-
atedcelldeathisinitiatedbytheligationofDR5withTRAILon
the cell surface.
TRAIL and DR5 have a great potential for cancer therapy.
For instance, we have reported potent antitumor effects of an
agonistic mAb against DR5 for the eradication of solid tumors
in mice (12–15). Recently, clinical trials with recombinant
TRAIL (dulanermin) and an agonistic anti-DR5 mAb (lexatu-
mumab) have been initiated against solid tumors, hematologi-
cal tumors, non-Hodgkin lymphoma, and non-small cell lung
cancer (7, 16). Some strategies to increase the cell surface
expression of DR5 on tumor cells are expected to improve the
efficacy of these agents.
Inthisstudy,wefoundthatlargeamountsofDR5werelocal-
ized in the nucleus in some tumor cell lines. We identified two
functional nuclear localization signal (NLS) sequences in DR5
recognized by importin (17, 18), the mutation of which abro-
gated the nuclear import of DR5. Moreover, the knockdown of
importin ?1 resulted in the accumulation of DR5 on the cell
surface and increased TRAIL/DR5-mediated apoptosis in
tumor cells. These findings may be translatable to the TRAIL
andanti-DR5mAbtherapiesbythetumor-specifictargetingof
importin ?1 using siRNA or a small molecule inhibitor of
importin-mediated nuclear transport.
EXPERIMENTAL PROCEDURES
Antibodies and Reagents—Mouse anti-human DR5 mAb
(HS201) and recombinant human TRAIL (rTRAIL) were pur-
chased from Alexis Biochemicals, and goat anti-human DR5
polyclonalantibody(pAb),controlrabbitIgG,controlgoatIgG,
and recombinant human IL-4 were from R&D Systems. Mouse
anti-tubulin mAb was from Sigma-Aldrich; rabbit anti-human
DR5 pAb (Ab-1) was from Calbiochem; and rabbit anti-human
YY1 pAb (C-20), rabbit anti-human importin ?1 pAb (H-300),
goat anti-human importin ?1 pAb (C-19), rabbit anti-STAT6
pAb (M-200), importin ?1 siRNA, and control siRNA were
obtained from Santa Cruz Biotechnology. Rabbit anti-human
histoneH3andmouseanti-humancaspase-8mAb(1C12)were
purchasedfromCellSignalingTechnology,andrabbitanti-hu-
man caspase-3 pAb was kindly provided by Dr. John C. Reed
(The Burnham Institute). Biotin-conjugated goat anti-mouse
IgG, HRP-conjugated rabbit anti-goat IgG, HRP-conjugated
goatanti-rabbitIgG,HRP-conjugatedstreptavidin,andcontrol
mouse IgG1 were from Dako Cytomation; HRP-conjugated
sheep anti-mouse IgG was from Amersham Biosciences/GE
Healthcare; and PE-conjugated goat anti-mouse IgG F(ab?)2
was from Caltag. Alexa Fluor 488-conjugated wheat germ
agglutinin(WGA),AlexaFluor488-conjugatedgoatanti-rabbit
IgG, and Alexa Fluor 594-conjugated streptavidin were
obtained from Invitrogen, and EZ-LinkTMNHS-Biotin (N-hy-
droxysuccinimide biotin) was purchased from Thermo
Scientific.
Cell Culture and Transfection of siRNA—A human hepato-
cellular carcinoma cell line (HepG2), a human cervical
adenocarcinoma cell line (HeLa), and a human prostate carci-
noma cell line (DU145) were obtained from ATCC and main-
tained in complete Dulbecco’s modified Eagle’s medium (Nis-
sui Pharmaceutical) supplemented with 10% heat-inactivated
fetal bovine serum, 2 mM L-glutamine, and 50 ?M 2-mercapto-
ethanol. For importin ?1 knockdown, 30 nM importin ?1
siRNA or control siRNA was transfected into HeLa cells using
Lipofectamine RNAiMAX (Invitrogen) according to the man-
ufacturer’s instructions. Cells were incubated for 24–48 h at
37 °C and then collected for subsequent analysis.
Measurement of TRAIL Sensitivity—Cells (1 ? 104in a vol-
ume of 100 ?l of culture medium/well) were seeded into a flat
bottomed 96-well plate (Corning) and incubated with various
concentrations of rTRAIL for 24 h at 37 °C. Cell viability was
determined by the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)[2H]tetrazolium monosodium salt (WST) assay
usingCellCountingKit-8(Dojindo)accordingtothemanufac-
turer’s instructions, and the percentage of cell death was calcu-
lated as described previously (19). To measure TRAIL sensitiv-
ity after importin ?1 knockdown, rTRAIL or vehicle alone was
added to the culture medium at 24 h post-transfection with
siRNA.
Flow Cytometry—To examine the cell surface expression of
DR5, 2 ? 105cells were incubated with 0.5 ?g of mouse anti-
humanDR5mAborisotype-matchedmouseIgG1for30minat
4 °C followed by PE-conjugated goat anti-mouse IgG F(ab?)2.
To detect the total cellular (namely cell surface and intracellu-
lar) expression of DR5, cells were treated with 70% ethanol in
PBSfor30minoniceandwashedwithPBSbeforethestaining.
The cells were then analyzed on a FACScanTM(BD Biosci-
ences), and the data were processed using the Cell Quest pro-
gram. The net mean fluorescence intensity was calculated as
described previously (20).
Immunostaining and Confocal Microscopy—After removal
of the culture medium, tumor cells incubated for 24–48 h at
37 °C on a poly-L-lysine-coated 4-well chamber slide (Nalge
Nunc) were rinsed in PBS and fixed with 8% paraformaldehyde
in 100 mM phosphate buffer for 30 min at 4 °C. After the
removal of the sidewall from the chamber slide, each chamber
was circled with a Dako pen, and the cells were treated with a
permeabilization buffer (Takara) according to manufacturer’s
instructions. The cells were incubated with appropriate con-
centrations of the primary antibodies or the isotype-matched
control Igs overnight at 4 °C for the detection of DR5 or for 1 h
at 37 °C for the detection of other proteins. Importin ?1 was
detected using Alexa Fluor 488-conjugated goat anti-rabbit
IgG,whereasDR5wasidentifiedbystainingwithbiotin-conju-
gated goat anti-mouse IgG and Alexa Fluor 594-conjugated
streptavidin. Endogenous biotin was blocked using an avidin-
biotinblockingkit(VectorLaboratories)accordingtotheman-
ufacturer’s specifications. Cell nuclei were counterstained with
Vectashield mounting medium for fluorescence with DAPI
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(Vector Laboratories) and viewed with a confocal microscope
(LSM510, Carl Zeiss).
Cell Fractionation and Western Blot Analysis—Cells were
lysed in NE-PER? nuclear and cytoplasmic extraction reagents
(Thermo Scientific) following the manufacturer’s instructions.
To prepare total cell lysate for the detection of importin ?1,
sample buffer containing SDS was added directly to the cells,
which were sonicated briefly. For the detection of caspases, the
cells were lysed in radioimmune precipitation assay buffer (50
mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 25 mM ?-glycerophosphate, 1 mM
sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, 1
mg/ml aprotinin, and 1 mg/ml leupeptin). The cytoplasmic,
nuclear, and total lysate fractions of the cells were subjected to
7.5, 10, or 16% SDS-PAGE under reducing conditions and
transferred onto PVDF membrane (Millipore). The mem-
braneswereanalyzedbyimmunoblottingwithappropriatepri-
mary antibodies followed by HRP-conjugated secondary anti-
bodies. The membranes were developed using an enhanced
chemiluminescence (ECL) Plus Western blotting detection kit
(GEHealthcare)orSuperSignalWestDuraExtendedDuration
Substrate (Thermo Scientific) according to the manufacturers’
instructions and were analyzed by LAS4000 (GE Healthcare).
ImmunoprecipitationandWesternblottingwereperformedas
described previously (21) on samples prepared from 5 ? 107
cells. For the detection of DR5, SDS-PAGE and Western blot-
tingwereperformedinnon-reducingconditions,andthemem-
branes were probed using biotin-conjugated goat anti-human
DR5 pAb followed by HRP-conjugated streptavidin.
Plasmid Construction and Transfection—The pEGFP-N1
vector was obtained from Clontech. The cDNA encoding the
DD-deleted DR5 containing the wild-type NLS sequences was
amplifiedbyPCRusingpMKITNeo-DR5(3)asatemplatewith
the following primers: sense, 5?-AATCTCGAGATGGAACA-
ACGGGGACAG-3?; and antisense, 5?-CGGAATTCGGGAG-
TCAAAGGGCACCAAGT-3?. This cDNA was designated
wild-typeNLS(WT-NLS).ToeliminatetheputativeNLSinthe
DR5 sequence, R15K16R17was mutated to AAA by using the
oligonucleotide sequences 5?-TCGGGGGCCGCGGCAGCG-
CACGGCCCA-3? and its reverse strand. This mutant was des-
ignated M1-NLS. A second NLS R322R323R324sequence was
mutated to AAA by using the oligonucleotide sequences 5?-
AGGTCTCAGGCGGCGGCGCTGCTGGTTCCA-3? and its
reverse strand. This mutant was designated M2-NLS. PCR
products were subcloned into the XhoI and EcoRI sites of the
pEGFP-N1 vector, and the constructs were verified by DNA
sequencing with an ABI PRISM 3100 sequence detection sys-
tem(AppliedBiosystems).Onedaybeforetransfection,2?105
cells were seeded on a 6-well plate and then transfected with 4
?g of plasmid DNA using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instructions. Following trans-
fection, the cells were cultured in selection medium containing
2 mg/ml G418, and GFP-positive cells were sorted using a
FACSCaliburTM(BD Biosciences).
RESULTS
Intracellular Localization of DR5 in Human Tumor Cells—
The detection of DR5 in tumor cell lines was achieved using
flow cytometry. As shown in Fig. 1A, HepG2 and HeLa cells
expressed a moderate level of DR5, whereas DU145 cells
expressed a high level of DR5 on their surface. After treatment
with 70% ethanol, which permeabilized the plasma membrane
and the nuclear membrane, an increase in DR5 was detected in
HepG2 and HeLa cells but not in DU145 cells, suggesting that
HepG2 and HeLa cells expressed intracellular DR5. To deter-
minetheintracellularlocalizationofDR5inthesecells,weper-
formed multicolor staining with anti-DR5 mAb, DAPI for
nuclear staining, and WGA for staining the Golgi apparatus
(22). As observed by confocal microscopy (Fig. 1B), a large
amount of DR5 was detected in the nuclei of both HepG2 and
HeLa cells. In contrast, the DR5 in DU145 cells was mostly
localized to the plasma membrane and the cytoplasm but was
barely detectable in the nucleus (Fig. 1B). To confirm the pres-
ence of DR5 in the nucleus of HeLa cells, we performed West-
ernblotanalysisofcytoplasmicandnuclearfractionsextracted
from HeLa cells. As shown in Fig. 1C, DR5 was clearly detected
in both the cytoplasmic and nuclear fractions of HeLa cells.
DR4 is another death receptor that is engaged by TRAIL like
DR5. We also investigated the expression and localization
of DR4 in tumor cell lines. As shown in supplemental Fig. 1A,
DU145 cells expressed a significant level of DR4 on the cell
surface,butHepG2andHeLacellsexpressedonlyalowlevelof
DR4. After the permeabilization of the plasma membrane and
thenuclearmembrane,theDR4levelsdidnotincreaseinanyof
the cell lines. Using confocal microscopy, DR4 was barely
detectableonthecellsurfaceorinthecytoplasmofHepG2and
HeLacells,whereasDR4waslocalizedonthecellsurfaceandin
the cytoplasm of DU145 cells (supplemental Fig. 2B). Nuclear
DR4 was not detectable in any of the cell lines.
CorrelationbetweenTRAILResistanceandNuclearLocaliza-
tion of DR5 in Tumor Cells—We next investigated the sensitiv-
ityofthesecelllinestoTRAIL.AsshowninFig.2A,DU145cells
were highly sensitive to TRAIL, and 25 ng/ml rTRAIL induced
80% cell death in 24 h. In contrast, the same doses of rTRAIL
had no effect on cell death in HepG2 and HeLa cells. We con-
firmed that concentrations up to 625 ng/ml rTRAIL did not
cause significant cell death in these cells even after 48 h (data
not shown). These results suggest that tumor cells containing
nuclearDR5areresistanttoTRAIL.Tofurthersubstantiatethe
TRAIL-inducedapoptosis,weperformedWesternblotanalysis
to estimate the cleavage of caspase-8 and caspase-3, which are
required for the generation of active initiator and executioner
caspases that induce apoptosis. As shown in Fig. 2B, in DU145
cells, procaspase-8 was completely cleaved, a significant
amountofprocaspase-3wascleavedfollowinga2-hincubation
with 125 ng/ml rTRAIL, and new bands corresponding to
cleaved caspase-8 and caspase-3 were detected. After a 24-h
incubationwithrTRAIL,procaspase-8wascleavedcompletely,
and only a small amount of procaspase-3 remained. In contrast
toDU145cells,onlysmallamountsofcleavedprocaspase-8and
cleaved procaspase-3 were detected in HeLa cells that were
incubated with rTRAIL for 2 h. After a 24-h incubation, there
was no difference in the expression levels of procaspase-8 and
procaspase-3 in the presence or absence of rTRAIL in HeLa
cells. Collectively, these data indicate that tumor cells contain-
ingnuclearDR5areresistanttoTRAIL-inducedapoptosis,and
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FIGURE1.CellsurfaceandnuclearlocalizationofDR5inHepG2,HeLa,andDU145cells.A,intactorpermeabilizedcellswerestainedwithanti-DR5mAb
orisotype-matchedcontrolmouseIgG1followedbyPE-conjugatedgoatanti-mouseIgG.Thestainedcellswereanalyzedbyflowcytometry.Boldlinesindicate
thestainingwithanti-DR5mAb;dottedlinesindicatebackgroundstainingwithcontrolmouseIgG1.B,tumorcellswerefixed,permeabilized,andstainedwith
Alexa Fluor 488-conjugated WGA and anti-DR5 mAb or isotype-matched control mouse IgG1 followed by biotin-conjugated goat anti-mouse IgG and Alexa
Fluor 594-conjugated streptavidin. Localization of DR5 was analyzed by confocal microscopy. White bars represent 20 ?m. C, cytoplasmic (C) and nuclear (N)
extracts from HeLa cells were subjected to Western blotting (WB) for DR5. Tubulin was used as a cytoplasmic marker, and YY1 was used as a nuclear marker.
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tumor cells having no nuclear DR5 are responsive to TRAIL-
induced apoptosis.
Association of DR5 with Importin ?1—The transport of pro-
teinsintothenucleusisgenerallymediatedbyimportinsviathe
recognition of NLS sequences. Analysis of the primary struc-
ture of human DR5 revealed two NLS-like sequences, RKR
starting at amino acid 15 and RRR starting at amino acid 322
(Fig. 3A, hDR5). Therefore, we investigated the association of
DR5 with importin ?1, which is essential for the transport of
various proteins into the nucleus regardless of the presence
of importin ? (18). Two-color immunostaining for DR5 and
importin ?1 showed that DR5 colocalized with importin ?1 in
the nucleus and in the perinuclear region of the cytoplasm in
HeLa cells (Fig. 3B). We next performed Western blot analysis
to detect importin ?1 in anti-DR5 immunoprecipitates from
HeLa cells and DU145 cells. As shown in Fig. 3C, importin ?1
was co-precipitated with DR5 from the HeLa cell lysate but not
from the DU145 cell lysate. We also detected DR5 in anti-im-
portin ?1 immunoprecipitates from the HeLa cell lysates (Fig.
3D). The results suggest that DR5 was transported into the
nucleus through its physical interaction with importin ?1 in
HeLa cells.
Identification of NLS within DR5—To identify the functional
NLS in human DR5, we generated two NLS mutants, M1-NLS
and M2-NLS, by replacing basic amino acids with AAA (Fig.
3A).InthesemutantsandintheWT-NLSconstructs,thedeath
domain was replaced with GFP to avoid selective death of the
transfectedcellsandtomonitortheexpressionoftheDR5-GFP
fusion proteins directly (Fig. 3A). After transfection of the con-
structsinpEGFP-N1vectorintoHeLacells,stabletransfectants
were generated. As estimated by cell surface staining of intact
cells with anti-DR5 mAb, the cell surface expression levels of
M1-NLS and M2-NLS correlated with the expression level of
theintroducedDR5-GFP(Fig.3E).Incontrast,cellsurfaceDR5
levelsdidnotincreaseinthemajorityofthecellsthatexpressed
DR5-GFPwithWT-NLS(Fig.3,EandF).Whencomparedwith
intact cells, the permeabilization of the plasma membrane and
nuclear membrane with 70% ethanol increased anti-DR5 mAb
staining in the WT-NLS cells but not in the M1-NLS or
M2-NLScells(Fig.3,FandG).Moreover,confocalmicroscopy
showed that the expression of DR5-GFP with WT-NLS was
mainlylocalizedinthenucleus,buttheexpressionofDR5-GFP
withthemutantM1-NLSorM2-NLSwaspredominantlylocal-
ized in the plasma membrane and the perinuclear region cor-
responding to the ER and Golgi apparatus (Fig. 3H). These
results indicate that both of the NLS-like sequences in human
DR5 are required for the nuclear import.
Knockdown of Importin ?1 Abrogates Nuclear Transport of
DR5—We next examined whether the localization of DR5 was
affected by the knockdown of importin ?1. When HeLa cells
were transfected with importin ?1 siRNA, importin ?1 protein
levels decreased substantially as estimated by Western blotting
(Fig. 4A). Compared with the control siRNA-treated cells,
which showed a prominent nuclear localization of DR5, confo-
cal microscopy of the importin ?1 siRNA-treated cells demon-
strated the distinct localization of DR5 in the WGA?Golgi
apparatus, the cytoplasm, and at the cell surface but not in the
nucleus (Fig. 4B). Similar results were obtained with the trans-
locationtoSTAT6,whichistranslocatedfromthecytoplasmto
the nucleus through the importin ?1 pathway after the stimu-
lationwithIL-4(23),whereashistoneH3,whichisknowntobe
importedtothenucleusbytransportin,importin?,importin5,
and importin 7 (24), was localized in the nucleus following
importin ?1 knockdown (Fig. 4B). Moreover, knockdown of
importin ?1 reduced the level of DR5 protein in the nuclear
fractionandincreasedtheDR5levelinthecytoplasmicfraction
ascomparedwiththecontrolsiRNA-treatedcells(Fig.4C).We
also investigated whether importin ?1 regulates the expression
and localization of DR4 as well as DR5. By flow cytometric and
confocal microscopic analyses, the expression level and local-
ization of DR4 were not affected by importin ?1 knockdown in
HepG2,HeLa,orDU145cells(supplementalFig.1,C,D,andE).
TheresultsindicatethatthenucleartransportisuniquetoDR5
and is mediated by importin ?1.
Surface Expression of DR5 Is Increased by Knockdown of
Importin?1—Weinvestigatedwhetherthecellsurfaceexpres-
sionofDR5increasesfollowingtheknockdownofimportin?1.
Flow cytometric analysis of intact or permeabilized cells after
anti-DR5 mAb staining showed that the cell surface expression
levels of DR5 on intact cells increased significantly following
transfection of importin ?1 siRNA compared with control
siRNA, whereas the total cellular DR5 expression levels in the
permeabilized cells were not affected (Fig. 4, D and E).
FIGURE 2. Sensitivity of HepG2, HeLa, and DU145 cells to TRAIL. A, tumor
cells (1 ? 104) were treated with various concentrations of rTRAIL (0–625
ng/ml) for 24 h. Cell death was determined by the WST assay as described
under “Experimental Procedures.” Data are expressed as the mean ? S.D. of
triplicatesamples.Similarresultswereobtainedinthreeindependentexper-
iments.B,1daybeforetheadditionofrTRAIL,HeLacells(2?105)andDU145
cells(5?105)wereseededinto6-wellplatesandtreatedwithorwithout125
ng/ml rTRAIL for, 2, 4, or 24 h. Whole cell extracts were subjected to Western
blotting for caspase-8, caspase-3, and tubulin, which was used as a marker.
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Knockdown of Importin ?1 Increases TRAIL Sensitivity of
TRAIL-resistant Cells—Finally, we examined whether the
knockdown of importin ?1 increases the sensitivity of tumor
cells to TRAIL. Phase-contrast microscopy showed that mas-
sive cell death was induced in the importin ?1 siRNA-treated
HeLa cells but not in the control siRNA-treated cells after a
24-h treatment with 25 ng/ml rTRAIL (Fig. 5A). WST assays
showed that the sensitivity of HeLa cells to either 25 or 125
ng/mlrTRAILincreasedfollowingthetransfectionofimportin
?1 siRNA compared with control siRNA (Fig. 5B). We also
examined the effect of importin ?1 knockdown on TRAIL sen-
sitivity in HepG2 and DU145 cells. As shown in supplemental
Fig. 2, A and B, TRAIL sensitivity of DU145 cells was not
affected by the knockdown of importin ?1. In contrast, the
TRAIL sensitivity of HepG2 cells was greatly increased by the
importin?1knockdown.Toconfirmtheinductionofapoptosis
by TRAIL following importin ?1 knockdown, we performed
Western blot analysis to estimate cleavage of caspase-8 and
caspase-3inwholecelllysatesfromHeLacells.Aftertheknock-
down of importin ?1, a large portion of the procaspase-8 and
procaspase-3 proteins was cleaved, and the cleaved forms were
clearly detected following a 4-h incubation with 125 ng/ml
rTRAIL (Fig. 5C). These data indicate that the TRAIL-induced
caspase-8andcaspase-3activationwasenhancedbytheimpor-
tin?1knockdown.BecauseDR4expressionwasnotaffectedby
importin ?1 knockdown (supplemental Fig. 1C), it is possible
that the increase in TRAIL sensitivity of HeLa and HepG2 cells
was caused by the augmentation of DR5-mediated apoptosis.
To confirm this possibility, we finally examined whether the
addition of anti-DR5 mAb inhibits the increase in TRAIL sen-
sitivity of HeLa cells. As expected, the increased sensitivity of
importin ?1 siRNA-treated cells to TRAIL was completely
abolished by the addition of an anti-DR5 blocking antibody
(Fig. 5D). These data indicate that the knockdown of importin
?1 can sensitize HeLa cells to TRAIL in a DR5-dependent
manner.
DISCUSSION
Inthisstudy,wefoundbymeansofconfocalmicroscopythat
DR5 was localized in the nucleus in HeLa and HepG2 cells.
PreviousreportshaveshownthatDR5localizespredominantly
in the plasma membrane and in the trans-Golgi network in
humanmelanomacells(25),melanocytes,endothelialcells,and
fibroblasts(26).Recently,DR5wasdetectedinthecytoplasmin
the majority of tissue samples obtained from patients suffering
from non-small cell lung carcinoma, and it was detected in the
nucleus in 27% of the same samples (27). Our present observa-
tionofthenuclearlocalizationofDR5inHepG2andHeLacells
is consistent with these clinical observations. It has also been
shownthatTRAILandDR5areinternalizedbyclathrin-depen-
dentand-independentendocytosis(1,28,29),andtheinternal-
ized DR5 is transported to the lysosome (30). However, our
study showed that DR5 did not colocalize with cathepsin D?
lysosomes in HeLa cells, HepG2 cells, or DU145 cells that lack
nuclear DR5 (data not shown). Moreover, the nuclear translo-
cation of DR5 was not observed even after preincubation with
rTRAIL (data not shown). Therefore, pathways other than
internalizationpathwaysmighttransportthenuclearDR5.Pro-
tein transport into the nucleus is generally mediated by the
importin system, which recognizes the NLS in the target pro-
teins (17, 18). We identified two functional NLS sequences in
DR5, the mutation of which abrogated the nuclear transport of
DR5 and confirmed the physical association of DR5 with
importin ?1. One NLS is located near the N terminus, corre-
sponding to the signal peptide, and the other NLS is located in
thecytoplasmicregionbetweenthetransmembraneregionand
the DD. Transmembrane and secreted proteins are generally
translocated across the ER membrane barrier through the
sequential actions of the signal recognition particle and its
receptor,whichisassociatedwithtranslocationintheERmem-
brane (31). The signal peptide is a 15–20-residue hydrophobic
N-terminalsignalsequencethatisrecognizedbythesignalrec-
ognition particle; however, no obvious sequence homologies
exist among signal peptides (32). Therefore, the signal pepti-
dasedoesnotrecognizeaspecificsequenceinthecleavagesite,
andnoteverytransmembraneorsecretedproteinisnecessarily
cleaved by signal peptidase (e.g. fibroblast growth factor
(FGF)-9 and FGF-16 (33, 34)). The molecular weights of mem-
brane-bound DR5 and nuclear DR5 were similar in our West-
ernblotanalysis,suggestingthatthesignalpeptidesequencein
DR5 is not targeted by a signal peptidase. This implies that the
N-terminalNLSofDR5isfunctional.Inapreviousstudyofthe
TNFreceptorfamily,CD40waslocalizednotonlyintheplasma
membrane and cytoplasm but also in the nucleus by the trans-
port through the NLS-importin pathway (35). To our knowl-
edge, our present study is the first to demonstrate the NLS-
mediated nuclear transport of a death receptor. Because the
ER-associated degradation system removes full-length trans-
membrane proteins from the ER into the cytoplasm (36), DR5
synthesized in the ER membrane might be released into the
cytoplasm where it is recognized by importin and transported
FIGURE3.AssociationofDR5withimportin?1andNLS-mediatednucleartransport.A,primarystructureoffull-lengthhumanDR5(hDR5),DD-deleted
DR5(WT-NLS),anditsN-orC-terminalNLSmutants(M1-NLSandM2-NLS).WT-NLS,M1-NLS,andM2-NLSwereC-terminallyfusedtoGFPinthepEGFP-N1
vector.TM,transmembraneregion.B,HeLacellswerefixed,permeabilized,andstainedwithrabbitanti-importin?1pAbandanti-DR5mAbasdescribed
under“ExperimentalProcedures.”ConfocalmicroscopywasperformedtovisualizethecolocalizationofDR5withimportin?1.Thewhitebarrepresents
10 ?m. C, DR5 protein interaction with importin ?1 in HeLa cells and DU145 cells was detected by immunoprecipitation (IP) with anti-DR5 mAb and
Westernblotting(WB)withrabbitanti-importin?1pAb.D,importin?1proteininteractionwithDR5inHeLacellswasdetectedbyimmunoprecipitation
(IP) with goat anti-importin ?1 pAb and Western blotting (WB) with anti-DR5 pAb as described under “Experimental Procedures.” E, intact HeLa cells
expressing WT-NLS, M1-NLS, or M2-NLS were stained with anti-DR5 mAb or control IgG1 followed by staining with PE-conjugated goat anti-mouse IgG.
GFP?cells and GFP?PE?cells were analyzed by flow cytometry. F, histogram data are shown for DR5-GFP?cells gated as indicated in E (R1, R2, and R3).
Boldlinesindicatestainingwithanti-DR5mAb;dottedlinesindicatebackgroundstainingwithcontrolmouseIgG1.Similarresultswereobtainedinthree
independent experiments. G, HeLa cells expressing WT-NLS, M1-NLS, or M2-NLS were permeabilized and stained with anti-DR5 mAb or control IgG1
followed by staining with PE-conjugated goat anti-mouse IgG. DR5-GFP?cells were analyzed by flow cytometry as described in E. Similar results were
obtained in three independent experiments. H, localization of WT-NLS, M1-NLS, or M2-NLS DR5-GFP fusion protein in HeLa cells was analyzed by
confocal microscopy. White bars represent 10 ?m.
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FIGURE4.Knockdownofimportin?1abrogatesnucleartransportofDR5andincreasescellsurfaceexpression.Importin?1siRNAorcontrolsiRNAwas
transfectedintoHeLacells.A,at48haftertransfection,totalcellularlysateswereanalyzedbyWesternblot(WB)forimportin?1andtubulin.B,cellswerealso
stainedwithAlexaFluor488-conjugatedWGAandanti-DR5mAb,anti-STAT6pAb,oranti-histoneH3pAbasdescribedinFig.1B.Toobservethetranslocation
ofSTAT6,thecellsweretreatedwith10ng/mlrecombinanthumanIL-4for45minbeforefixation.TheintracellulardistributionofDR5,STAT6,andhistoneH3
was determined by confocal microscopy. White bars represent 10 ?m. C, proteins in cytoplasmic (C) and nuclear (N) fractions were analyzed by Western blot
(WB) for DR5, tubulin, and histone H3. D, intact or permeabilized cells were stained with anti-DR5 mAb or isotype-matched control IgG1 followed by PE-
conjugated goat anti-mouse IgG. Staining was analyzed by flow cytometry as described in Fig. 1A. Similar results were obtained in three independent
experiments. E, net mean fluorescence intensity (MFI) was calculated from the data in D as described under “Experimental Procedures.” Data are the mean ?
S.D. of three independent experiments. *, p ? 0.05 by Student’s t test.
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intothenucleus.Inthisscenario,DR5isnottranslocatedtothe
Golgi apparatus nor is it modified with glycans. Although DR5
does not contain any potential N-linked glycosylation sites (5),
the modification of death receptors by O-glycosylation is very
important in cancer cells (1, 37). Previously, it has been
reported that the O-glycosylation of DR4 and DR5 in cancer
cells modulates the sensitivity of cells to TRAIL by promoting
ligand-induced receptor clustering and subsequent caspase-8
activation (37). Given that protein modification by O-linked
glycosylation can occur in the nucleus (38), DR5 that is local-
izedinthenucleuscouldbemodifiedbyO-glycosylation.How-
ever,itisunlikelythatnuclearDR5wouldbetransportedtothe
plasmamembrane.Atpresent,itremainsunclearwhyaportion
of DR5 is translocated to the plasma membrane and another
portion of DR5 is transported to the nucleus. Further studies
are needed to determine how the localization of DR5 is
regulated.
TumorcellsexpressingDR5inthenucleus,suchasHeLaand
HepG2cells,exhibitahighresistancetoTRAILcomparedwith
thetumorcellsthatlacknuclearDR5,suchasDU145cells.The
nuclear localization of DR5 is thought to be advantageous for
tumor cells to escape from TRAIL/DR5-mediated cell death.
FIGURE 5. Knockdown of importin ?1 increases DR5-mediated sensitivity of HeLa cells to rTRAIL. A, importin ?1 siRNA or control siRNA was
transfectedintoHeLacells(5?103)ina96-wellplate.At24hpost-transfection,cellsweretreatedwithorwithout25ng/mlrTRAILfor24handobserved
using a phase-contrast microscope. Scale bars indicate 50 ?m. B, at 24 h after transfection with importin ?1 siRNA or control siRNA, the indicated
concentrations of rTRAIL were added. Cell death was quantified by WST assay as described under “Experimental Procedures.” Data are the mean ? S.D.
of triplicate samples. **, p ? 0.02; ***, p ? 0.001 by Student’s t test. Similar results were obtained in three independent experiments. C, 1 day before
transfection, HeLa cells (1 ? 105) were seeded onto a 6-well plate and transfected with importin ?1 siRNA or control siRNA. After a 24-h incubation, cells
were treated with or without 125 ng/ml rTRAIL for 4 or 24 h. Whole cell extracts were subjected to Western blot for caspase-8, caspase-3, importin ?1,
andtubulin.D,cellswerealsoincubatedwithanti-DR5mAborisotype-matchedcontrolmouseIgG1for30minat37 °Cbeforetheadditionof125ng/ml
rTRAIL. After a 24-h incubation, cell death was analyzed by WST assay. Data are the mean ? S.D. of triplicates. **, p ? 0.01 by Student’s t test. Similar
results were obtained in three independent experiments.
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Therefore,onecouldhypothesizethatthenuclearDR5contrib-
utes to the survival of tumor cells. The knockdown of importin
?1 resulted in a significant sensitization of HeLa and HepG2
cells to TRAIL/DR5-mediated cell death by enhancing the cell
surface expression of DR5. Thus, the nuclear localization of
DR5 can limit the DR5/TRAIL-induced cell death of tumor
cells. Kau et al. (39) pointed out the mislocalization from the
cytoplasm to the nucleus of some proteins in cancers (e.g.
nuclear factor-?B and ?-catenin) and reported that protein
mislocalization resulted in cancer cell intervention. Our obser-
vations correlate with these in vivo findings. Moreover, it has
been shown that nuclear CD40 binds to and stimulates the B
lymphocyte stimulator/B cell activation factor promoter and
regulates the growth and survival of B lymphocytes (35). In this
context,futurestudieswilldeterminewhethernuclearDR5has
a similar function in tumor cell survival. The regulation of sen-
sitivity and resistance to TRAIL-induced cell death has been
investigated in many reports. For example, it has been sug-
gestedthattherelativeexpressionofdeathreceptorsanddecoy
receptors that lack a DD may regulate TRAIL sensitivity (40,
41). Other factors such as TRAIL-induced nuclear factor-?B
activation and death inhibitors including cellular FADD-like
interleukin-1? converting enzyme inhibitory protein and
X-linked inhibitor of apoptosis protein have also been impli-
cated in the differential sensitivity to TRAIL (1, 2, 11, 42). On
the other hand, many cancer therapeutic drugs have been
shown to enhance the TRAIL sensitivity of tumor cells by up-
regulating DR4 or DR5 expression (6, 7). We have demon-
stratedthattheknockdownofimportin?1efficientlysensitized
TRAIL-resistant tumor cells to rTRAIL by increasing the cell
surface expression of DR5. siRNA and shRNA have been
reported to be effective in silencing the gene in vivo, and phase
I–III clinical studies have already been initiated against various
diseases including cancers (43). Therefore, tumor-specific
delivery of importin ?1 siRNA or a recently identified small
moleculeinhibitorofimportin?-mediatednuclearimport(44)
may be useful in applications that will improve the antitumor
effectsofrecombinantTRAIL(dulanermin)andanti-DR5mAb
(lexatumumab) for the therapeutic treatment of human
cancers.
Acknowledgments—We deeply thank Drs. Nobuhiko Kayagaki and
NorikoKoyamaforprovidingpMKITNeo-DR5vector;AkemiKoyan-
agi, Keiko Maeda, Hisaya Akiba, and Toshiro Takai for technical
assistance; Tamami Sakanishi for cell sorting; and Sachiko Kom-
azawa-Sakon for DNA sequencing.
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1
Supplemental Data

Importin ? ?1-mediated nuclear localization of death receptor 5 (DR5) limits DR5/TNF-related
apoptosis inducing ligand (TRAIL)-induced cell death of human tumor cells
Yuko Kojima, Masafumi Nakayama, Takashi Nishina, Hiroyasu Nakano, Makoto Koyanagi,
Kazuyoshi Takeda, Ko Okumura, and Hideo Yagita

Supplemental Materials and Methods

Mouse anti-human DR4 mAb (HS101) was purchased from Alexis Biochemicals. For importin
?1 knockdown, 30 nM importin ?1 siRNA or control siRNA were transfected into HepG2 cells using
Lipofectamine RNAiMAX (Invitrogen), and transfected into DU145 cells using Lipofectamine 2000
(Invitrogen), respectively, according to the manufacturer’s instructions.

Supplemental Figure Legends

Supplemental FIGURE 1. Cell surface and intracellular localization of DR4 in HepG2, HeLa,
and DU145 cells. A. Intact or permeabilized cells were stained with anti-DR4 mAb or
isotype-matched control mouse IgG1, followed by PE-conjugated goat anti-mouse IgG. The staining
was analyzed by flow cytometry. Bold lines indicate staining with anti-DR4 mAb; dotted lines
indicate background staining with control mouse IgG1. B. Tumor cells were fixed, permeabilized, and
stained with Alexa 488-conjugated WGA and anti-DR4 mAb or isotype-matched control mouse IgG1,
followed by biotin-conjugated goat anti-mouse IgG and Alexa 594-conjugated streptavidin.
Localization of DR4 was analyzed by confocal microscopy. White bars represent 10 ?m. C. At 48 h
after transfection with importin ?1 siRNA or control siRNA, intact or permeabilized cells were
stained with anti-DR4 mAb or isotype-matched control IgG1, followed by PE-conjugated anti-mouse
IgG Ab. Staining was analyzed by flow cytometry as described in Supplemental Fig. 1A. Similar
results were obtained in three independent experiments. D. At 48 h after transfection with importin ?1
siRNA or control siRNA, total cellular lysates prepared from HepG2 and DU145 cells were analyzed
by Western blotting (WB) for importin ?1 and tubulin. E. After the transfection with importin ?1
siRNA or control siRNA, DU145 cells were stained with Alexa 488-conjugated WGA and anti-DR4
mAb as described in Supplemental Fig. 1B. The intracellular distribution of DR4 was determined by
confocal microscopy. White bars indicate 10 ?m.

Supplemental FIGURE 2. Knockdown of importin ? ?1 increases TRAIL sensitivity of HepG2
cells, but not DU145 cells. A. Importin ?1 siRNA or control siRNA were transfected into HepG2 and
DU145 cells (1 ? 104) on 96-well plate. At 24 h post-transfection, cells were treated with or without 5
ng/mL rTRAIL for 24 h, and were observed using a phase-contrast microscope. Scale bars represent
50 ?m. B. At 24 h after transfection with importin ?1 siRNA or control siRNA, indicated
concentrations of rTRAIL were added. Cell death was quantified by WST assay as described in
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2
Experimental Procedures. Data are the mean ± SD of triplicate samples. * P < 0.02, ** P < 0.01 by
Student’s t-test. Similar results were obtained in three independent experiments.
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Kojima Supplemental FIGURE 1.
A
HepG2 HeLa DU145
Intact
Permeabilized
DR4 expression
Cell number
HeLa
B
Merge
WGA
DR4
DAPI
Merge
WGA
DR4
DAPI
HepG2 DU145
Merge
DR4
DAPI WGA
Importin ? ?1 siRNA
Control siRNA
DR4
DR4
WGA
WGA
DAPI
DAPI
Merge
Merge
E
C
Importin ? ?1
siRNA
Control
siRNA
Permeabilized
Intact
DR4 expression
Cell number
Permeabilized Intact
DU145 HeLa
HepG2
Permeabilized Intact
D
Tubulin
Importin ? ?1
Control
IgG
Anti-Importin ? ?1
or Tubulin
Control
siRNA
Importin ? ?1
siRNA
Control
siRNA
WB:
Tubulin
Importin ? ?1
DU145
HepG2
DU145
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Kojima Supplemental FIGURE 2.
Control siRNA
Importin ? ?1 siRNA
rTRAIL (ng/mL)
Cell death (%)
1 5
25
0
10
20
30
40
50
B
rTRAIL (ng/mL)
Cell death (%)
1 5 25
0
10
20
30
40
50
60
70
HepG2 DU145
**
*
*
A
rTRAIL (+)
rTRAIL (-)
Importin ? ?1 siRNA Control siRNA
HepG2 DU145
Importin ? ?1 siRNA Control siRNA
at JUNTENDO UNIVERSITY, on November 29, 2012
www.jbc.org
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