Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-kappaB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-kappaB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

Full text

MOLECULAR AND CELLULAR BIOLOGY, Feb. 2003, p. 777–790 Vol. 23, No. 3
0270-7306/03/$08.00⫹0 DOI: 10.1128/MCB.23.3.777–790.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Proteasome Inhibition Results in TRAIL Sensitization of Primary
Keratinocytes by Removing the Resistance-Mediating Block
of Effector Caspase Maturation†
Martin Leverkus,
1
* Martin R. Sprick,
2
Tina Wachter,
1
Thilo Mengling,
1
Bernd Baumann,
3
Edgar Serfling,
4
Eva-B. Bro¨cker,
1
Matthias Goebeler,
1
Manfred Neumann,
4
and Henning Walczak
2
Department of Dermatology
1
and Institute of Pathology,
4
University of Wu¨rzburg Medical School, 97080 Wu¨rzburg,
Division of Apoptosis Regulation, Tumor Immunology Program, German Cancer Research Center, 69120
Heidelberg,
2
and Department of Physiological Chemistry, University of Ulm,
89081 Ulm,
3
Germany
Received 22 May 2002/Returned for modification 18 July 2002/Accepted 6 November 2002
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against
transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhi-
bition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-␬B activation. Here
we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is
removed by proteasome inhibitors independent of NF-␬B in primary human keratinocytes. This sensitization
was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further
downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways
were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are
needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 matu-
ration. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but
not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased
resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are
potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby
complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.
Apoptotic cell death is an important biological process that
is required to maintain the integrity and homeostasis of mul-
ticellular organisms. Inappropriate or impaired apoptosis has
been implicated in the development of many human diseases,
including cancer (71). The death-inducing members of the
tumor necrosis factor (TNF) family, TNF, CD95/APO-1/Fas
ligand (CD95L), and TNF-related apoptosis-inducing ligand
(TRAIL/APO-2L), have been studied most intensively. These
studies have resulted in elucidation of their role in activation-
induced cell death, autoimmune disorders, immune privilege,
and tumor evasion from the immune system (reviewed in ref-
erences 79, 82, and 85).
TRAIL has attracted attention for its ability to preferentially
kill tumor cells while most normal cells were resistant both in
vitro (56, 86) and in vivo (1, 20, 80). The functional analysis of
the TRAIL receptor-ligand system has been complicated by
the fact that a total of five different receptors for this cytokine
has been identified (reviewed in reference 46). Although the
major role of TRAIL is the induction of apoptosis, it has also
been shown in earlier overexpression studies to activate other
signaling pathways, such as the transcription factor NF-␬B (11,
62). NF-␬B is known to induce genes involved in apoptosis
resistance. Inhibition of NF-␬B can sensitize cells for TNF␣-or
TRAIL-induced apoptosis, depending on the cell type, sug-
gesting that distinct signaling pathways modulate the effect of
TRAIL in a cell type-specific manner (2, 27, 76).
The early biochemical events resulting in apoptosis induc-
tion by ligand-induced death receptor cross-linking have been
studied by the analysis of the so-called death-inducing signal-
ing complex (DISC) (33, 81). Cross-linking of CD95 or the two
apoptosis-inducing TRAIL receptors (TRAIL-R1 and
TRAIL-R2) results in the recruitment of Fas-associated death
domain (FADD; also called MORT1) and caspase 8 to the
DISC (3, 34, 68). In a homotypic interaction, the death domain
of FADD binds to the death domain of CD95. The death
effector domain of FADD in turn interacts with the death
effector domain of procaspase 8 and thereby recruits this
proenzyme to the DISC (51). Procaspase 8 is proteolytically
cleaved and thereby activated at the DISC. Activated caspase
8 then initiates the apoptosis-executing caspase cascade (81).
This cascade is further controlled by “cross talk” between the
intrinsic (mitochondrial) and extrinsic (death receptor) cell
death pathways, thereby modulating the outcome of death
receptor triggering (58). In that respect it has been shown for
CD95L and recently also for TRAIL (24, 41) that its proapo-
ptotic signaling can be blocked by Bcl-2 or Bcl-X
L
overexpres
-
sion in some cell types, whereas other cell types cannot be
protected by overexpression of these molecules, leading to the
* Corresponding author. Mailing address: Department of Derma-
tology, University of Wu¨rzburg Medical School, Josef-Schneider-Str. 2,
97080 Wu¨rzburg, Germany. Phone: 49 931 201 26356. Fax: 49 931 201
26700. E-mail: leverkus_m@klinik.uni-wuerzburg.de.
† This article is dedicated to Harald zur Hansen on the occasion of
his retirement as head of the German Cancer Research Center (Deut-
sches Krebs forschungszentrum) in Heidelberg with gratitude and ap-
preciation for 20 years of leadership.
777

concept of two different cell types utilizing distinct signaling
pathways with or without the necessity for mitochondrial con-
tribution (60).
Further complexity is added to the regulatory pathways in-
volved in death receptor sensitivity by proteins that are capable
of inhibiting active caspases. These proteins are called inhibi-
tor-of-apoptosis proteins (IAPs) (14, 25). IAPs are a family of
proteins defined by baculovirus repeat (BIR) domains and, in
some cases, a zinc ring finger domain (14, 25). IAPs like X-
linked IAP (XIAP), Livin/MLIAP, cIAP1, and cIAP2 block
apoptosis by directly inhibiting caspases. For some IAPs, an
involvement in caspase-independent pathways of apoptosis was
postulated (14, 65). XIAP is the most potent inhibitor of
caspases among the above-mentioned IAPs (14).
The ubiquitin-proteasome pathway plays a central role in
the regulation of essential cellular processes such as cell cycle
control, transcription, signal transduction, and apoptosis (28,
36). Many key regulatory proteins are controlled by ubiquiti-
nation, which targets them for degradation by the 26S protea-
some (29, 39). A recent report suggested that proteasome
inhibitors are able to induce apoptosis in transformed but not
in normal lymphocytes (50). Well-known proteasomal targets
include the NF-␬B/I␬B system, p53, and IAPs (28). Earlier
studies showed that NF-␬B activation is efficiently blocked by
proteasome inhibitors (27). These data support the view that
sensitization to TNF␣- or TRAIL-induced apoptosis by pro-
teasome inhibitors is explained by the inhibition of NF-␬B
transcriptional activity (27, 30).
The mechanisms responsible for the resistance of primary
cells to TRAIL are not fully understood. Although initial re-
ports suggested that differential expression of TRAIL-R3 and
TRAIL-R4 might determine resistance to TRAIL in primary
cells, later studies demonstrated the important contribution of
intracellular regulatory pathways in primary cells (42). To fur-
ther investigate the resistance to TRAIL in primary human
keratinocytes, we analyzed proteasome-mediated resistance to
TRAIL-induced apoptosis and its neutralization by protea-
some inhibitors. We show that inhibition of proteasomal func-
tion effectively sensitizes primary keratinocytes to TRAIL in-
dependent of NF-␬B and downstream of the TRAIL DISC.
This sensitization required the activation of mitochondrial sig-
naling pathways necessary for XIAP inhibition. Our data sug-
gest that one important step to overcome the intracellular
resistance of primary keratinocytes to TRAIL is the inhibition
of XIAP by activation of mitochondrial apoptosis signaling
pathways.
MATERIALS AND METHODS
Materials. The protease inhibitor z-Val-Ala-Asp-fluoromethyl ketone (zVAD-
FMK) was obtained from Bachem (Heidelberg, Germany). If not indicated
otherwise, all other reagents were of reagent grade and obtained from Serva
(Heidelberg, Germany). The following antibodies were used: poly(ADP-ribose)
polymerase (G. Poirier, CHUL Research Center, Quebec, Canada), caspase 3
(MF 393 and MF 397), and XIAP (MF 478; gifts from D. Nicholson, Merck
Frosst Corp, Quebec, Canada); Smac (kindly provided by M. MacFarlane, Le-
icester, United Kingdom); and HtrA2/Omi (generously provided by R. Taka-
hashi, RIKEN Brain Science Institute, Saitama, Japan). The FLICE (C-15) (51)
and cFLIP (NF-6) (61) monoclonal antibodies were kindly provided by P. H.
Krammer and are available from Alexis (Gruenberg, Germany). Monoclonal
antibodies against FADD and XIAP were purchased from Transduction Labo-
ratories (San Diego, Calif.), caspase 10 monoclonal antibodies (clone 4C1) were
from MBL International (Watertown, Mass.), cytochrome c monoclonal anti-
bodies were from Pharmingen (San Diego, Calif.), antibodies to I␬B␣ (C-21)
were from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), and antibodies to
IKK2 (antibody 2684) were from New England Biolabs, Boston, Mass.
The monoclonal antibodies specific for the different TRAIL receptors were
generated by immunizing mice with TRAIL receptor-Fc fusion proteins. The
specificity of the monoclonal antibodies was confirmed by staining cells trans-
fected with expression plasmids for TRAIL-R1 to TRAIL-R4 (H. Walczak,
unpublished observation). Anti-TRAIL-R1 (clone HS101) and anti-TRAIL-R2
(clone HS201), both of the murine immunoglobulin G1 (IgG1) isotype, were
used for fluorescence-activated cell sorting staining. The monoclonal antibodies
are also available from Alexis (Gruenberg, Germany). Recombinant leucine
zipper (LZ)-TRAIL was generated as described before (78), and Flag-TRAIL
was expressed as previously described (32). Horseradish peroxidase-tagged don-
key anti-rabbit immunoglobulin antibodies and horseradish peroxidase-tagged
goat anti-mouse IgG were from Pharmingen (Hamburg, Germany), and horse-
radish peroxidase-conjugated goat anti-mouse IgG1 and IgG2b were obtained
from Southern Biotechnology Associates (Birmingham, Ala.).
Tissue culture. Primary keratinocytes were prepared from neonatal foreskins
as previously described (42). Cells were kept in serum-free KGM medium (Cell-
systems, St. Katharinen, Germany). Cells were only used up to passage 3 for all
experiments. The spontaneously transformed keratinocyte line HaCaT was
kindly provided by N. Fusenig (German Cancer Research Center, Heidelberg,
Germany) and cultured as described before (4).
Subfractionation of cytoplasmic proteins. Cytosolic extracts were generated
with a modified digitonin lysis protocol (66). Following trypsinization, cells were
washed with phosphate-buffered saline, permeabilized with digitonin (Fluka,
Taufkirchen, Germany) at a concentration of 150 ␮g/ml for 10 min, and centri-
fuged at 5,000 ⫻ g for 5 min to remove cellular debris. The resulting supernatants
were spun again at 13,000 ⫻ g for 30 min at 4°C. Equal amounts of supernatant
were analyzed by Western blot. Blots were subsequently rehybridized with mono-
clonal antibodies to cytochrome c oxidase subunit IV (Molecular Probes Inc.,
Eugene, Oreg.) to control for mitochondrial lysis (data not shown).
Western blot analysis. Total cellular proteins were collected as described
before (44) with the exception that complete protease inhibitor cocktail (Roche
Molecular Diagnostics, Mannheim, Germany) was used. From 20 to 75 ␮gof
protein was electrophoresed on sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes.
Blocking of membranes and incubation with the indicated primary and appro-
priate secondary antibodies were performed essentially as described elsewhere
(43). Bands were visualized with the ECL detection kit (Amersham).
Analysis of death-inducing signaling complex. For precipitation of the native
TRAIL DISC, 5 ⫻ 10
6
human primary keratinocytes were used for each condi
-
tion. Cells were washed once with RPMI medium at 37°C and subsequently
incubated for the indicated time periods at 37°C in the presence of 1 ␮gof
Flag-TRAIL per ml precomplexed with 2 ␮g of anti-Flag antibody M2 (Sigma,
Taufkirchen, Germany) per ml for 15 min or, for the unstimulated control, in the
absence of Flag-TRAIL. DISC formation was stopped by washing the monolayer
twice with ice-cold phosphate-buffered saline. Cells were lysed on ice by addition
of 1 ml of lysis buffer (30 mM Tris-HCl [pH 7.5] at 21°C, 120 mM NaCl, 10%
glycerol, 1% Triton X-100, complete protease inhibitor cocktail [Roche Molec-
ular Diagnostics, Mannheim, Germany]). After 15 min of lysis, the lysates were
centrifuged at 50,000 ⫻ g for 15 min to pellet cellular debris. DISC complexes
were precipitated from the lysates by coincubation with 20 ␮l of protein G beads
(Roche, Mannheim, Germany) for 12 h on an end-over-end shaker at 4°C.
For precipitation of the nonstimulated receptors, 200 ng of Flag-TRAIL and
400 ng of anti-Flag M2 were added to the lysates prepared from nonstimulated
cells at 200 ng to control for protein association with a nonstimulated recep-
tor(s). Ligand affinity precipitates were washed five times with lysis buffer before
the protein complexes were eluted from the beads by addition of 15 ␮lof2⫻
standard reducing sample buffer. Subsequently, proteins were separated by SDS-
PAGE on 4 to 12% NuPage Bis-Tris gradient gels (Invitrogen, Karlsruhe, Ger-
many) before detection of DISC components by Western blot analysis.
Determination of DEVDase activity. Cells were lysed following treatment in
buffer containing 50 mM HEPES-KOH (pH 7.0), 2 mM EDTA, 10% sucrose,
0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS),
and 5 mM dithiothreitol. Equal amounts of protein were incubated with 2,5 ␮M
biotinylated DEVD-acyloxymethyl ketone (biotin-DEVD-AOMK) (kindly pro-
vided by D. Nicholson, Merck Frosst Corp, Quebec, Canada) for 30 min at 37°C
(57). Biotin-DEVD-AOMK is an irreversible caspase inhibitor that covalently
binds to DEVD-cleaving caspases, resulting in the biotin labeling of active
caspase fragments (57, 72). Samples were separated by SDS-PAGE and trans-
ferred to nitrocellulose membranes. Following overnight incubation with perox-
idase-conjugated streptavidin (Dako Diagnostika, Hamburg, Germany), biotin-
778 LEVERKUS ET AL. MOL.CELL.BIOL.

labeled fragments were visualized with the ECL detection kit (Amersham,
Arlington Heights, Ill.). Membranes were subsequently stripped and reprobed
with antibodies to caspase 3 (MF 393) to detect caspase 3-specific fragments.
Coimmunoprecipitation of XIAP. Primary and transformed keratinocytes (2 ⫻
10
6
cells) were treated with 250 ng of TRAIL per ml for 3 h and lysed in TENT
buffer (50 mM Tris [pH 8.0], 2 mM EDTA, 150 mM NaCl, and 1% Triton X-100)
on ice. Equal amounts of cellular proteins were precipitated with 1 ␮g of either
rabbit IgG, rabbit caspase 3 (MF 397), or XIAP (MF 478) antiserum (kindly
provided by D. Nicholson, Merck Frosst Corp, Quebec, Canada) in the presence
of 20 ␮l of protein A-Sepharose beads overnight. Precipitates were washed four
times with lysis buffer before the protein complexes were eluted from the beads
by addition of 2⫻ standard reducing sample buffer. Subsequently, proteins were
separated by SDS-PAGE before detection of XIAP by Western blot analysis with
monoclonal antibodies to XIAP (Transduction Laboratories, San Diego, Calif.).
FACScan analysis. For analysis of the mitochondrial transmembrane potential
(⌬⌿m), 5 ⫻ 10
5
cells were resuspended in Dulbecco’s modified Eagle’s medium
containing 40 nM tetramethylrhodamine ethyl ester (59) and incubated at 37°C
for 15 min as described before (21). After being washed once with ice-cold
phosphate-buffered saline, cells were immediately analyzed. For surface staining
of TRAIL-R1 and TRAIL-R2, 2 ⫻ 10
5
cells were detached by incubation with
phosphate-buffered saline containing 5 mM EDTA, followed by brief trypsiniza-
tion, and incubated with monoclonal antibodies against TRAIL-R1 (HS101),
TRAIL-R2 (HS201), or control murine IgG1 at 10 ␮g/ml, followed by biotinyl-
ated secondary goat anti-mouse immunoglobulin antibodies and phycoerythrin-
conjugated streptavidin (Pharmingen, Hamburg, Germany). For all experiments,
10
4
cells were analyzed by FACScan (Becton Dickinson & Co, San Jose, Calif.).
Apoptosis and cytotoxicity assays. Crystal violet staining of surviving attached
cells was performed 16 to 24 h after addition of LZ-TRAIL in 24-well plates (1
⫻ 10
5
cells) or 96-well plates (2 ⫻ 10
4
cells) as described before (42). Parallel
plates were examined by propidium iodide staining and flow cytometric analysis
of subdiploid DNA content as described before (42). For annexin-propidium
iodide staining, 2 ⫻ 10
5
to 5 ⫻ 10
5
cells were stained with 10 ␮l of annexin
V-fluorescein isothiocyanate conjugate (Becton Dickinson & Co, San Jose, Cal-
if.) and 5 ␮l of propidium iodide (50 ng/ml) according to the manufacturer’s
instructions, incubated for 15 min at room temperature in the dark, and imme-
diately analyzed.
Electrophoretic mobility shift assays. Nuclear and cytoplasmic extracts of
primary keratinocytes were prepared according to Schreiber et al. (63) with slight
modifications as previously described (53). The oligonucleotide probe used was
a consensus NF-␬B binding site derived from the ␬B element of the murinein-
terleukin-2 promoter (TCEd
A⬎C
) (6): 3⬘-GACCAAGAGGGATTTCACCCCT
AAATC-5⬘. The following antibodies were used for the supershift electro-
phoretic mobility shift assay: anti-p65-NF-␬B (Santa Cruz Biotechnology Inc.,
Santa Cruz, Calif.; A, sc-109), anti-c-Rel (Santa Cruz; N, sc-070), anti-RelB
(Santa Cruz; C-19, sc-226), anti-p50-NF-␬B (Santa Cruz; NLS, sc-114), and
anti-p52-NF-␬B (Upstate Biotechnology Inc., Lake Placid, N.Y.; 06-413).
Retroviral infection. The pCFG5-IEGZ retroviral vector containing inserts of
the transdominant ⌱␬B␣ (⌱␬B␣-TD) mutant or kinase-dead IKK2 (IKK2-KD)
mutant was used for infection of primary human keratinocytes as described
earlier (13) with the exception that viral supernatants were generated in KGM.
Infection efficiency of keratinocytes ranged from 30% to 80%, as determined by
green fluorescent protein fluorescence 72 h after infection. At this time point,
keratinocytes were used for cytotoxicity assays and biochemical characterization.
Transient-transfection assay for XIAP. Cells were transfected with the
Nucleofector technology (Amaxa, Ko¨ln, Germany) according to the recom-
mended protocols. Briefly, 2 ⫻ 10
6
transformed keratinocytes were resuspended
in 100 ␮l of Cell Line R solution. Then 3 ␮g of pEBB-XIAP vector or pEBB
control vector (kindly provided by Colin Duckett, University of Michigan Med-
ical School) was mixed with the suspension, added to an Amaxa cuvette, and
nucleofected with program U27. Cells were then diluted with 500 ␮l of warm
RPMI and added to six-well plates containing 1 ml of warm medium. After a 10-h
incubation period at 37°C, biochemical and functional studies were performed as
indicated in the figure legends. Transfection efficiency, as determined by green
fluorescent protein expression in parallel plates, varied between 50% and 70%.
RESULTS
Primary keratinocytes are sensitized to TRAIL-induced apo-
ptosis by proteasome inhibition. We have previously shown
that primary keratinocytes are relatively resistant to TRAIL-
induced apoptosis compared to transformed keratinocytes
(42). Inhibition of proteasome function modulates apoptotic
signaling pathways in a variety of tumor cells (16, 28). We thus
explored the potential role of proteasomal function for the
resistance of primary keratinocytes to TRAIL-mediated apo-
ptosis. To this end, we used MG115 as a proteasomal inhibitor
in our studies. MG115 treatment resulted in a dramatic sensi-
tization of primary keratinocytes for TRAIL-induced apoptosis
in a concentration-dependent manner (Fig. 1) (55). Moreover,
two other known proteasome inhibitors, lactacystin (19) and
epoxomicin (52), similarly sensitized primary keratinocytes for
TRAIL-induced apoptosis (data not shown). The broad-spec-
trum caspase inhibitor zVAD-FMK completely abolished
TRAIL-induced apoptosis under these conditions, confirming
the critical role of caspases in this process (Fig. 1). Our data
suggest that peptide aldehyde inhibitors such as MG115 inter-
fere with proteasome-regulated signaling pathways, resulting
FIG. 1. Proteasome inhibitor MG115 sensitizes primary keratino-
cytes to TRAIL-induced apoptosis. (A) Primary keratinocytes were
treated with 50 ng of TRAIL per ml for 16 h. Caspase-dependent
cytotoxicity of TRAIL (sub-G
0
DNA analysis) is only detectable in the
presence of the proteasome inhibitor MG115 (10 ␮M). zVAD-FMK
(40 ␮M) completely abolishes TRAIL-induced apoptosis. (B) Dose-
dependent effect of MG115 on TRAIL-induced cell death in primary
keratinocytes was determined by crystal violet staining 16 h after ad-
dition of the indicated concentrations of TRAIL.
V
OL. 23, 2003 EFFECTOR CASPASE INHIBITION IN PRIMARY KERATINOCYTES 779

in increased sensitivity of primary keratinocytes to TRAIL-
induced apoptosis.
TRAIL induces NF-␬B activation in primary keratinocytes.
One important target of proteasomal degradation is the I␬B/
NF-␬B system (29, 48). NF-␬B has potent antiapoptotic prop-
erties, particularly in TNF␣-induced apoptosis (30), whereas
its role in TRAIL-induced apoptosis is more controversial (26,
27). To study whether the increased sensitivity to TRAIL-
induced apoptosis may be mediated by inhibition of NF-␬B
function, we first analyzed TRAIL-induced NF-␬B activation
in primary keratinocytes and the impact of the proteasome
inhibitor MG115. Employing electrophoretic mobility shift as-
says, we detected an increase in NF-␬B DNA binding activity
upon stimulation with TRAIL at concentrations of between 50
and 500 ng/ml (Fig. 2A). This induction was observed within 30
min and increased further up to 6 h after stimulation (Fig. 2B).
Although zVAD-FMK has been shown to completely block
TRAIL-induced apoptosis (42), it did not negatively affect
TRAIL-induced NF-␬B activation (Fig. 2B), indicating that
caspase activation is not required for TRAIL-induced NF-␬B
activation. The supershift electrophoretic mobility shift assay
demonstrated that TRAIL-induced NF-␬B binding activity
consisted mainly of p50 homodimers as well as p65/p50 het-
erodimers. Nevertheless, weak supershift signals of p65/c-Rel
complexes were also detectable (Fig. 2C). In line with reports
for other cell types (27), TRAIL-induced NF-␬B DNA binding
activity and degradation of I␬B␣ were dose-dependently inhib-
ited by the proteasome inhibitor MG115 (Fig. 2D).
NF-␬B activation does not modulate TRAIL sensitivity in
keratinocytes. The proteasome has been shown to modulate
multiple signaling pathways, including NF-␬B, p53, and IAPs
(9, 88). In order to inhibit NF-␬B more specifically, we next
studied whether ectopic expression of dominant negative mu-
tants of IKK2 (IKK2-KD) or ⌱␬B␣ (⌱␬B␣-TD) influenced
FIG. 2. TRAIL dose-dependently enhances NF-␬B binding in primary keratinocytes. (A) Primary keratinocytes were treated with 5 to 1,000
ng of TRAIL per ml for 6 h and analyzed for NF-␬B DNA binding by electrophoretic mobility shift assay with nuclear extracts. Induction of p65/p50
heterodimers (upper arrow) or p50 homodimers (lower arrow) is shown. As a control for the specificity of DNA binding, control experiments with
an excess of unlabeled probe were performed (not shown). Cell viability was determined in parallel experiments by crystal violet staining and is
indicated as a percentage of the control value. (B) Primary keratinocytes were exposed to 250 ng of TRAIL per ml for6hinthepresence or
absence of 40 ␮M zVAD-FMK. Survival of cells was determined in parallel experiments as described for A. (C) Composition of TRAIL-induced
nuclear NF-␬B complexes in primary keratinocytes. Following incubation of primary keratinocytes with TRAIL (250 ng/ml) for 4 h, supershift
electrophoretic mobility shift analysis of inducible NF-␬B-specific complexes was performed with nuclear extracts. Each of the Rel-specific
antibodies (1 ␮l of antibody [Ab]; see the text) was added to nuclear protein extracts. Control supershift electrophoretic mobility shift assays
(EMSAs) were performed with normal rabbit serum (not shown). Arrows indicate supershifted complexes. (D) MG115 leads to dose-dependent
inhibition of TRAIL-induced NF-␬B activation (upper panel) and I␬B␣ degradation (lower panel). Membranes were rehybridized with antitubulin
monoclonal antibodies to confirm comparable loading of cytoplasmic proteins.
780 LEVERKUS ET AL. M
OL.CELL.BIOL.

TRAIL sensitivity in keratinocytes. We thus examined primary
keratinocytes following retroviral infection with either IKK2-
KD, ⌱␬B␣-TD, or a viral control construct. Similar to the data
shown in Fig. 2D, overexpression of these proteins efficiently
inhibited TRAIL-induced degradation of ⌱␬B␣ and NF-␬B
activation compared to vector-transfected control cells (Fig.
3A and data not shown). However, sensitivity to TRAIL-in-
duced apoptosis was not significantly modulated by these mu-
tants (Fig. 3B).
Consistent with these data, neither TRAIL nor the protea-
some inhibitor MG115 modulated mRNA levels of antiapo-
ptosis genes such as cIAP-1, cIAP-2, TRAF-1, or TRAF-2 in
keratinocytes when assayed by an RNase protection assay (not
shown). Taken together, our results indicate that neither
TRAIL-induced NF-␬B activation nor constitutive NF-␬B ac-
tivation significantly modulates resistance to TRAIL. More
importantly, our data suggest that the peptide aldehyde inhib-
itor MG115 interferes with other proteasome-regulated signal-
ing pathways which finally result in increased sensitivity of
keratinocytes to TRAIL-induced apoptosis.
Sensitization to TRAIL-induced apoptosis by proteasome
inhibition is mediated downstream of the death-inducing sig-
naling complex. We next determined whether proteasome in-
hibitors modulate surface expression of the proapoptotic
TRAIL-R1 and TRAIL-R2. As shown in Fig. 4A, exposure of
primary keratinocytes to MG115 did not significantly modulate
surface expression of either TRAIL-R1 or TRAIL-R2, which
are both expressed in primary keratinocytes. TRAIL stimula-
tion of these receptors leads to rapid activation of caspase 8
and, as recently shown, also of caspase 10 (35, 67, 84) at the
death-inducing signaling complex (DISC) initiating the
caspase cascade (3, 34, 68). Thus, increased caspase 8 or 10
recruitment to and/or subsequent activation at the DISC could
be responsible for the observed sensitization to TRAIL-in-
duced apoptosis following MG115 treatment.
We therefore examined whether MG115 modifies the acti-
vation of caspase 8 or 10 at the DISC by immunoprecipitation
of the native TRAIL DISC from primary keratinocytes (Fig.
4B). DISC analysis demonstrated rapid recruitment and cleav-
age of caspases 8 and 10 at the DISC. The structurally related
protein cFLIP, which is also expressed in primary keratino-
cytes, acts as an inhibitor of CD95L- and TRAIL-induced
apoptosis by inhibiting the activation of caspase 8 at the DISC
(38, 42, 61). In line with our previous observation (42), large
amounts of cFLIP
L
and cFLIP
S
(where L indicates long and S
indicates short) were found in the DISC. Importantly, the
DISC composition remained unchanged after treatment with
the proteasome inhibitor MG115 (Fig. 4B). Thus, neither
DISC activation nor surface expression of the TRAIL-R pro-
teins explains the increased sensitivity to TRAIL of primary
keratinocytes treated with MG115. However, it is currently not
known whether recruitment and activation of caspases is the
sole predictor of the activity of DISC constituents. Therefore,
we next examined activation kinetics of the known DISC-as-
sociated proteins in cellular lysates of primary keratinocytes in
the presence and absence of MG115.
Indeed, TRAIL induced rapid cleavage of caspase 8 to
p43/41 as well as to the active p18 fragment, which were de-
tectable within 60 to 120 min of stimulation. Furthermore,
rapid cleavage of cFLIP
L
(p43) was seen within 60 to 120 min.
Similarly, specific cleavage of caspase 10 to the p47/p43 inter-
mediates as well as to a 25-kDa fragment representing the fully
cleaved prodomain was detectable (Fig. 5). Slight differences in
the amount of the active fragment of caspase 8 (p18) or the
fully cleaved prodomain of caspase 10 (p25) were observed at
later time points in the presence of the inhibitor MG115.
Addition of zVAD-FMK fully inhibited TRAIL-induced cleav-
age of caspase 8, caspase 10, and, to a lesser extent, cFLIP
L
(Fig. 5). In the presence of MG115, levels of cFLIP
S
were
increased compared to the barely detectable levels found un-
der control conditions.
These data show that MG115-induced sensitization of pri-
mary keratinocytes to TRAIL is not regulated at the level of
caspase 8 or 10 activation. Collectively, our findings indicate
FIG. 3. Overexpression of dominant negative mutant of IKK2
(IKK2-KD) or transdominant I␬B␣ (I␬B␣-TD) inhibits TRAIL-in-
duced degradation of I␬B␣ without modulation of TRAIL-induced
apoptosis. (A) Primary human keratinocytes were retrovirally trans-
duced with IKK2-KD, I␬B␣-TD, or control vector and subsequently
analyzed for expression of mutant proteins as well as degradation of
endogenous I␬B␣ following stimulation with 500 ng of TRAIL per ml
for1hbyWestern blotting. Membranes were rehybridized with anti-
tubulin monoclonal antibodies to confirm comparable loading of cel-
lular proteins. (B) Primary keratinocytes overexpressing IKK2-KD or
I␬B␣-TD as described for A were either left untreated or incubated
with 25, 50, or 125 ng of LZ-TRAIL per ml for 6 h. Thereafter, the
percentage of dead green fluorescent protein-positive cells was deter-
mined by tetramethylrhodamine ethyl ester staining. Data are shown
as mean ⫾ standard deviation of two independent experiments.
V
OL. 23, 2003 EFFECTOR CASPASE INHIBITION IN PRIMARY KERATINOCYTES 781

that the dramatic sensitization of primary keratinocytes for
TRAIL-mediated apoptosis induced by proteasome inhibition
is not due to differential activation of the apoptotic signaling
pathway at the TRAIL DISC. Thus, this sensitization of pri-
mary keratinocytes for TRAIL-induced apoptosis must be reg-
ulated at a step downstream of the DISC.
Autocatalytic maturation of caspase 3 to active enzyme is
blocked in primary keratinocytes. Caspase 3 is the major ef-
fector caspase in most cell types (54). The activation of caspase
3 is tightly regulated at several levels, beginning with a proteo-
lytic cut between the large and small subunits, followed by
removal of the prodomain. While the first step occurs mainly
via active initiator caspases such as caspase 8, 9, or 10, the
second step is mediated by autoproteolytic maturation of
caspase 3 (54). In order to further examine the level at which
MG115 acts to sensitize primary keratinocytes for TRAIL-
induced apoptosis, we examined the activation pattern of
caspase 3. Interestingly, TRAIL stimulation of primary kera-
tinocytes in the absence of MG115 resulted in the cleavage of
caspase 3 to a 20-kDa fragment within 60 to 120 min without
detectable induction of apoptosis. However, full processing to
the 17-kDa fragment was detectable when cells were treated
with TRAIL in the presence of MG115 (Fig. 6A).
To further examine the activity of caspase 3 under these
conditions, we used biotinylation of catalytically active caspase
FIG. 4. Caspases 8 and 10 are recruited and cleaved at the native
TRAIL DISC independent of MG115. (A) Primary keratinocytes were
treated with 10 ␮M MG115 or diluent for 6 h and subsequently ana-
lyzed for surface expression of TRAIL-R1 and TRAIL-R2 by fluores-
cence-activated cell sorting. MG115 does not modulate TRAIL-R1 or
TRAIL-R2 surface expression on primary keratinocytes. (B) Analysis
of the native TRAIL DISC in primary keratinocytes. Cells were
treated for the indicated times with MG115 or diluent alone. The
stimulated TRAIL DISC (⫹) or unstimulated receptors (⫺) were
precipitated with Flag-TRAIL precomplexed with 2 ␮g of anti-Flag
antibodies per ml (clone M2). The resulting protein complexes were
separated by SDS-PAGE and analyzed by Western blot for compo-
nents of the TRAIL DISC. The adaptor protein FADD was detectable
only when stimulated receptors were precipitated. Also, recruitment of
the two isoforms of caspase 8, caspase 8a and 8b, was detected only
following stimulation of TRAIL receptors. In addition, the intermedi-
ate cleavage products, p43/p41, of the two isoforms of caspase 8 were
detectable in the DISC. For caspase 10, the full-length forms caspase
10d (p59) and caspase 10a (p55) as well as the cleavage products
p47/43 and the p25 fragment, corresponding to the prodomain (Pro-
dom.), were detectable in the immunoprecipitates. Analysis of cFLIP
levels revealed that both isoforms, cFLIP
L
and cFLIP
S
, were present in
the TRAIL DISC, with comparable levels in control cells and MG115-
treated cells. For cFLIP
L
, only the p43 cleavage product was detectable
in the DISC, indicative of proteolytic processing by active caspases in
the DISC. The migration position of the IgG heavy and light chains of
the precipitating anti-Flag M2 antibody as well as that of protein G
cross-reacting with the antibody used for Western blot detection
(caspase 10, lower part) are indicated by asterisks.
FIG. 5. Proteasome inhibitor MG115 does not influence early
TRAIL-induced caspase 8, caspase 10, and cFLIP cleavage. Primary
keratinocytes were incubated for 60 min with 10 ␮M MG115 or diluent
alone and subsequently treated with 50 ng of TRAIL per ml for the
indicated times. TRAIL treatment led to cleavage of caspase 8 (p43/
p41/p18), caspase 10 (p47/43/25), and cFLIP (p43) under both condi-
tions, as determined by Western blot analysis. Membranes were rehy-
bridized with antitubulin monoclonal antibodies to confirm comparable
loading of proteins.
782 LEVERKUS ET AL. M
OL.CELL.BIOL.

3 in cellular lysates of primary keratinocytes (57). This tech-
nique allows the simultaneous detection of enzymatic activity
and molecular weight, enabling the parallel monitoring of cleav-
age and activity of caspase 3. Biotin-labeled fragments of
caspase 3 were clearly detectable in lysates of cells treated with
TRAIL in the presence of MG115. Interestingly, in cells
treated with TRAIL alone, full-length caspase 3 was marginally
labeled, indicating some enzymatic activity of the full-length
caspase 3, confirming the recent report by Roy et al. (57) (Fig.
6B, upper panel). Consistent with the data shown in Fig. 6A,
reprobing of the same membrane with caspase 3-specific anti-
bodies revealed the comparable presence of the cleaved p20
fragment of caspase 3 in lysates treated with TRAIL alone
(Fig. 6B, middle panel). These data demonstrate that the ini-
tial cleavage of caspase 3 is not blocked in TRAIL-treated
primary keratinocytes but that the resulting cleavage product
was enzymatically inactive. In line with this observation, cleav-
age of poly(ADP-ribose) polymerase, a known substrate of
caspase 3 (40), was only detected when caspase 3 was further
processed to the p17 fragment after treatment with MG115
(Fig. 6B, lower panel). Taken together, our findings indicate
that TRAIL-induced full processing of caspase 3 to the active
17-kDa fragment which is required for full enzymatic activity is
blocked in primary human keratinocytes and that this block is
released upon treatment of primary keratinocytes with the
proteasome inhibitor MG115.
Differential expression of XIAP in primary versus trans-
formed keratinocytes. In order to further investigate the inter-
play between caspase 3 maturation and TRAIL receptor trig-
gering, we compared two parameters of TRAIL-induced
apoptosis in primary versus transformed keratinocytes: the
dose-dependent cleavage pattern of caspase 3 and induction of
apoptosis as determined by hypodiploidy analysis (Fig. 7). In
line with our previous findings (42), low concentrations of
TRAIL were sufficient to induce significant apoptosis in trans-
formed keratinocytes, whereas primary keratinocytes were
dose-dependently protected but nonetheless succumbed to
high concentrations of TRAIL (Fig. 7). In contrast to the
findings in primary keratinocytes (Fig. 7, lower panel, left
lanes), the p20 fragment of caspase 3 was hardly detectable in
transformed keratinocytes, whereas the p17 and a p15 frag-
ment of caspase 3 were readily detected in these cells when
stimulated with low concentrations of TRAIL (Fig. 7, lower
panel, right lanes). Full cleavage correlated with detectable
activity of effector caspases, as examined by affinity labeling
(Fig. 7, upper panel). Interestingly, high concentrations of
TRAIL sufficient to induce partial apoptosis led to labeling of
full-length caspase 3 in primary keratinocytes (Fig. 7, upper
panel).
Taken together, these data pointed to a potential inhibitor
of effector caspases blocking the full maturation of cleaved
caspase 3 in primary keratinocytes. XIAP, the most potent
caspase inhibitor of the IAP family, has been shown to selec-
tively inhibit the cleaved forms of caspase 3 and 7 (14). We
therefore examined the expression level of XIAP in primary
and transformed keratinocytes. Intriguingly, Western blot
analysis revealed that the 57-kDa XIAP protein was present in
primary but not in transformed keratinocytes (Fig. 8A, lysate).
Furthermore, a 29-kDa fragment of XIAP generated by
caspase-mediated cleavage (15) was only detected in TRAIL-
FIG. 6. Full processing of caspase 3 to a 17-kDa fragment in
TRAIL-treated primary keratinocytes upon proteasome inhibition.
(A) Primary keratinocytes were incubated for 60 min with 10 ␮M
MG115 (right part) or diluent alone (left part) and subsequently
treated with 50 ng of TRAIL per ml for the indicated time intervals.
TRAIL leads to substantial cleavage of caspase 3 to a 20-kDa fragment
(p20), whereas only in the presence of the proteasome inhibitor is a
17-kDa fragment (p17) detectable. No cleavage fragments were de-
tected in the presence of zVAD-FMK or after incubation with MG115
alone. Membranes were rehybridized with antitubulin monoclonal an-
tibodies to confirm comparable loading of proteins. (B) TRAIL-in-
duced effector caspase activity with affinity labeling with biotinylated
DEVD-AOMK (see Materials and Methods). Activity of cleaved ef-
fector caspases by 50 ng of TRAIL per ml requires the presence of the
proteasome inhibitor MG115. Primary keratinocytes were treated as
above, and DEVD-specific caspase activity in cellular lysates was an-
alyzed 3 h after TRAIL stimulation. DEVDase activity of cleaved
fragments (p17/p15, upper panel) was only detectable in cells treated
with TRAIL in the presence of MG115, whereas TRAIL or MG115
alone did not induce detectable caspase activity. Slight labeling of
full-length caspase 3 was detected in cells treated with TRAIL alone.
Subsequent rehybridization of the blot with caspase 3-specific antibody
confirmed that the slower-migrating caspase 3 species (p20) did not
have detectable enzymatic activity in primary keratinocytes (middle
panel). In line, poly(ADP-ribose) polymerase (PARP) cleavage was
only detected upon treatment with TRAIL in the presence of MG115
(lower panel). Membranes were rehybridized with antitubulin mono-
clonal antibodies to confirm comparable loading of proteins.
V
OL. 23, 2003 EFFECTOR CASPASE INHIBITION IN PRIMARY KERATINOCYTES 783

treated primary keratinocytes. These data suggest that XIAP
may act as an inhibitor of TRAIL-induced apoptosis in primary
keratinocytes by blocking the full cleavage and, thus, the ac-
tivity of caspase 3.
In order to investigate further the interaction of caspase 3
with XIAP, immunoprecipitation studies were performed.
While antibodies to XIAP readily precipitated XIAP in un-
treated as well as TRAIL-treated primary keratinocytes, coim-
munoprecipitation of XIAP with caspase 3 was only detected
following TRAIL treatment (Fig. 8A). These data demonstrate
the interaction of caspase 3 with XIAP in TRAIL-treated pri-
mary keratinocytes under physiological conditions.
In order to address the question of whether the lack of XIAP
in transformed keratinocytes contributes to the high TRAIL
sensitivity of these cells, transient-transfection studies were
performed. Following overexpression of XIAP in transformed
keratinocytes (Fig. 8B, middle lanes) resistance to TRAIL-
induced apoptosis was increased (Fig. 8C). Furthermore, in-
creased levels of caspase 3 p32 and p20 were detected follow-
ing TRAIL treatment in XIAP-overexpressing cells.
Interestingly, high levels of overexpressed XIAP (Fig. 8B, up-
per panel) did not completely block TRAIL-induced caspase 3
maturation compared to primary keratinocytes (Fig. 8B, lower
panel). Because the transfection efficiency in these experi-
ments was between 50 and 70%, the remaining detectable fully
cleaved caspase 3 (Fig. 8B) as well as the partial apoptosis
protection in functional assays (Fig. 8C) might, at least in part,
be explained by the percentage of cells without efficient XIAP
expression. However, we cannot exclude that additional differ-
ences in the inhibition of effector caspase activation exist be-
tween primary and transformed keratinocytes. Future studies
are required to delineate this point. Taken together, our data
demonstrate that the lack of XIAP contributes to the higher
sensitivity of transformed keratinocytes to TRAIL.
We next investigated whether the expression level or cleav-
age pattern of XIAP following TRAIL treatment of primary
keratinocytes is modulated by MG115. We therefore examined
XIAP levels during TRAIL treatment in the presence and
absence of MG115. Limited cleavage of XIAP to a 29-kDa
fragment was detected in the presence and absence of MG115.
However, a slight increase in cleaved XIAP was detected at
later time points in MG115-treated cells (Fig. 8D). This in-
crease correlated with full caspase 3 processing (compare Fig.
6A), suggesting that XIAP cleavage is a secondary event me-
diated by active caspase 3. Importantly, MG115 treatment
alone did not significantly change XIAP levels (Fig. 8D). These
data provide evidence that the mechanism of MG115-induced
increase of TRAIL sensitivity does not involve cleavage of
XIAP or modulation of XIAP expression but rather direct
inhibition of caspase 3 by XIAP.
MG115 sensitizes primary keratinocytes to TRAIL-induced
apoptosis upstream of or at the mitochondrial level. Apoptotic
activation of the mitochondrion results in the release of pro-
teins such as cytochrome c, Smac/DIABLO, and HtrA2/Omi
from the intermitochondrial membrane space (49, 73). Smac/
DIABLO and HtrA2/Omi release from mitochondria in turn
leads to caspase activation by inhibiting the antiapoptotic func-
tion of several IAPs, particularly XIAP (8, 17, 18, 45, 74). Our
finding that XIAP is highly expressed in primary keratinocytes
prompted us to investigate whether proteasome inhibition may
allow TRAIL-induced release of Smac/DIABLO or HtrA2/
Omi from mitochondria, resulting in XIAP inhibition.
FIG. 7. Caspase 3 activation pattern in primary and transformed keratinocytes. (A) DEVD-specific caspase activity in cellular lysates was
analyzed 3 h after TRAIL stimulation in primary keratinocytes (PK) and transformed HaCaT keratinocytes (TK). DEVDase activity of p19, p17,
and p15 (upper panel) was detectable in transformed keratinocytes treated with TRAIL, whereas the same concentrations of TRAIL did not
induce detectable DEVDase activity of cleaved fragments of caspase 3 in primary keratinocytes. Induction of apoptosis was monitored in parallel
plates by hypodiploidy analysis. Higher concentrations of TRAIL sufficient to induce partial cell death in primary keratinocytes led to a
dose-dependent increase in full-length caspase 3 labeling, indicative of some enzymatic activity of full-length caspase 3. Subsequent reprobing of
blots with caspase 3-specific antibody confirmed that low concentrations of TRAIL were sufficient to induce the slower-migrating caspase 3 form
(p20) in primary keratinocytes. In contrast, mainly the mature cleaved forms p17 and a smaller fragment (p15) were detected in transformed
keratinocytes, even at low concentrations of TRAIL. Ponceau Red staining of the membrane confirmed comparable loading of proteins. HRP,
horseradish peroxidase.
784 LEVERKUS ET AL. M
OL.CELL.BIOL.

FIG. 8. XIAP confers resistance to TRAIL-induced apoptosis in primary keratinocytes. (A) XIAP is expressed in primary keratinocytes (PK)
but not in transformed keratinocytes (TK) and can be coimmunoprecipitated under native conditions with caspase 3 following TRAIL treatment.
Total cellular lysates from primary keratinocytes and transformed keratinocytes were prepared, and equal amounts of protein were either analyzed
by Western blotting (25 ␮g) with XIAP monoclonal antibodies (lysate, left and right side) or subjected to immunoprecipitation (500 ␮g). A 57-kDa
protein representing full-length XIAP was detected in primary (right) but not transformed (left) keratinocytes. An additional band of roughly 65
kDa (asterisks) detected in total lysates was shown to be a nonspecific band (immunoprecipitation: XIAP, right lanes). Following immunopre-
cipitation with caspase 3 antiserum, full-length but not cleaved XIAP was detected only in immunoprecipitates of TRAIL-treated primary
keratinocytes PK (immunoprecipitation: caspase 3). (B) Transfection of transformed keratinocytes with XIAP leads to decreased cleavage of
full-length caspase 3 and increased detection of caspase 3 p20. Transformed keratinocytes were transiently transfected with 3 ␮g of pEBB-XIAP
vector or pEBB control vector as indicated in Materials and Methods. Then 125 ng of TRAIL per ml was added to the six-well plates, and cellular
lysates were collected 3 h later. Primary keratinocytes were treated in parallel plates. The same membrane was first incubated with monoclonal
antibodies to XIAP and subsequently reprobed with caspase 3 antibodies. Rehybridization with antitubulin monoclonal antibodies confirmed
comparable loading of cellular proteins. Shown is a representative experiment of a total of three independent experiments. (C) XIAP overex-
pression leads to relative resistance of transformed keratinocytes to TRAIL-induced apoptosis. Cells were transfected as described for B and
treated with the indicated concentrations of TRAIL, and viability was determined 6 h later with annexin/propidium iodide staining. Shown are
means and standard deviations for two independent experiments. (D) Primary keratinocytes were incubated for 60 min with 10 ␮M MG115 (right
part) or diluent alone (left part) and subsequently treated with 50 ng of TRAIL per ml for the indicated time periods. TRAIL treatment resulted
in cleavage of XIAP to a 29-kDa fragment (p29), with similar cleavage at early time points in the presence or absence of the proteasome inhibitor.
No cleavage fragments were detected in the presence of zVAD-FMK or after incubation with MG115 alone for 4 h, at which time XIAP levels
were comparable to those found under control conditions.
V
OL. 23, 2003 EFFECTOR CASPASE INHIBITION IN PRIMARY KERATINOCYTES 785

We therefore examined the kinetics of the release of mito-
chondrial proteins into the cytosol during MG115-induced sen-
sitization of primary keratinocytes to TRAIL. In cells treated
with TRAIL alone, release of cytochrome c, Smac/DIABLO,
or HtrA2/Omi could not be detected (Fig. 9A). In contrast, in
cells treated with TRAIL in the presence of MG115, cyto-
chrome c, Smac/DIABLO, and HtrA2/Omi were rapidly re-
leased from the mitochondria, whereas exposure to MG115
alone had no effect (Fig. 9A). Consistently, the quantitative
disruption of the inner mitochondrial membrane potential
(⌬␺
m
) which represents an important “point of no return” of
apoptosis (37), was detected in TRAIL-treated primary kera-
tinocytes in the presence but not in the absence of MG115
(Fig. 9B). Taken together, these data suggest that proteasome
inhibition sensitizes primary keratinocytes for TRAIL-induced
apoptosis by disabling the XIAP-mediated inhibition of auto-
proteolytic maturation of caspase 3 by allowing Smac/DIA-
BLO and HtrA2/Omi release from the mitochondria and, thus,
inhibition of XIAP.
DISCUSSION
Primary keratinocytes are resistant to TRAIL concentra-
tions that induce apoptosis in transformed keratinocytes but
nonetheless succumb to high levels of TRAIL (42). This ob-
servation led us to further explore intracellular signaling path-
ways in primary keratinocytes which might modulate sensitivity
to death ligands such as TRAIL (26, 27, 76). One important
step for the regulation of such signaling pathways is the pro-
grammed degradation of specific proteins via the proteasome
(16). Our initial finding that proteasome inhibition dramati-
cally sensitized primary cells to TRAIL led us to examine a
prime candidate for the regulation of TRAIL sensitivity, the
transcription factor NF-␬B. Several reports have suggested
that TNF␣-mediated apoptosis in particular is regulated by a
group of genes induced by NF-␬B, including cIAP-1, cIAP-2,
TRAF-1, and TRAF-2 (83, 87). Regarding TRAIL, conflicting
data have been published (26, 27), suggesting that for this
death ligand, cell type-specific differences may exist. Here we
show that TRAIL induced a moderate but reproducible acti-
vation of NF-␬B-specific DNA binding and I␬␤␣ degradation
under nonoverexpression conditions of TRAIL-R proteins.
Our data are in line with recent reports utilizing 293 and HeLa
cell lines (23, 27, 76). However, specific blockade of NF-␬Bby
ectopically expressed inhibitors of NF-␬B activation (i.e., IKK2
or I␬␤␣) failed to sensitize keratinocytes to TRAIL. These
data clearly indicate that other signaling pathways modulated
by proteasome function play a more important role for sensi-
tivity to TRAIL, at least in keratinocytes.
TRAIL-induced signaling pathways have thus far been stud-
ied mainly in cell lines that are highly sensitive to TRAIL or in
cell lines that are resistant to receptor ligation due to artificial
overexpression of intracellular inhibitors such as cFLIP (31).
In order to study the possible involvement of TRAIL DISC
formation in the mechanism of TRAIL resistance of primary
keratinocytes, we first had to elucidate the composition of the
native TRAIL DISC in these cells. We determined that the
TRAIL DISC in primary keratinocytes contains FADD,
caspase 8, caspase 10, the second initiator caspase recently
shown to be recruited to the native TRAIL DISC in several cell
lines (35, 67, 84), cFLIP
L
, and cFLIP
S
. Earlier overexpression
studies have demonstrated that cFLIP
L
is capable of inhibiting
TRAIL-induced apoptosis in keratinocytes (42) and melanoma
cells (22). Detailed comparative and kinetic studies of the
proteolytic activity of the TRAIL DISC are required to deter-
mine the exact contribution of all DISC components in these
cells. Nonetheless, our studies clearly showed that in primary
keratinocytes, a cell type that expresses TRAIL-R1 and
TRAIL-R2 at high levels, inhibition of the proteasome did not
modulate the recruitment or cleavage of FADD, cFLIP, and
FIG. 9. MG115 sensitizes primary keratinocytes upstream or at the
mitochondrial level. (A) Cytochrome c, Smac/DIABLO, and HtrA2/
Omi release after TRAIL treatment of primary keratinocytes. Cells
were treated for the indicated time intervals with 50 ng of TRAIL per
ml in the presence or absence of 10 ␮M MG115. Cytoplasmic lysates
were subsequently prepared as described in Materials and Methods
and analyzed for cytochrome c, Smac/DIABLO, and HtrA2/Omi by
Western blot analysis. Equal loading of cytoplasmic proteins was de-
termined by reprobing of blots with antitubulin antibodies.
(B) TRAIL-induced loss of mitochondrial transmembrane potential
⌬⌿
m
requires the presence of the proteasome inhibitor MG115. Cells
were incubated for 60 min with either diluent alone or 10 ␮M MG115,
followed by treatment with 50 ng of TRAIL per ml. Cells were ana-
lyzed for ⌬⌿
m
by staining with 40 nM tetramethylrhodamine ethyl
ester 4 h later. Quantitative disruption of ⌬⌿
m
was only seen in cells
treated with TRAIL in the presence of the proteasome inhibitor
MG115.
786 LEVERKUS ET AL. M
OL.CELL.BIOL.

caspase 8 or 10 at the DISC, suggesting that the proteasome
inhibitor MG115 sensitizes primary keratinocytes downstream
of the TRAIL DISC. Intriguingly, active initiator caspases can
be detected in TRAIL-treated primary keratinocytes without
significant apoptosis. Cleavage of caspase 3 to the 20-kDa
fragment is found at similar levels in cells treated with TRAIL
in the absence versus the presence of proteasome inhibitors.
These data further support the notion that caspase 8 is suffi-
ciently activated to achieve initial cleavage of caspase 3. Thus,
our data indicate that besides the activity of the TRAIL DISC,
additional intracellular regulatory mechanisms exist. These
mechanisms control TRAIL-induced effector caspase activity
before a “point of no return” is passed, at which apoptosis
induction is inevitable. Importantly, in primary cells, as exem-
plified here for primary keratinocytes, low levels of initiator
caspase activation might be tolerated without induction of apo-
ptosis, thereby activating nonapoptotic signaling pathways.
To date, little is known about the signaling events associated
with TRAIL receptor ligation downstream of the DISC. In
particular, the role of mitochondrial signaling pathways re-
mains to be elucidated for TRAIL-induced apoptosis. In some
cell lines examined, TRAIL-induced apoptosis was shown to
be largely independent of mitochondrial signaling pathways
(32, 77). In other studies, Bcl-x
L
protected pancreatic carci
-
noma cells from TRAIL-induced apoptosis (24), and inactiva-
tion of Bax was recently shown to confer resistance to TRAIL-
induced apoptosis in colon cancer lines (41). Taken together,
these data support the view that cell type-specific differences
downstream of active initiator caspases might be important for
the cell fate decision following stimulation of TRAIL recep-
tors.
Our studies show that proteasome inhibitors induce major
and early differences in the TRAIL-dependent release of cy-
tochrome c, Smac/DIABLO, and HtrA2 (64) despite similar
early activation of initiator caspases. Importantly, the protea-
some inhibitor alone had no effect on mitochondrial cyto-
chrome c, Smac/DIABLO, or HtrA2 release which is in con-
trast to findings obtained in transformed cell lines (75). This
difference might be due to a variable sensitivity of tumor cells
to proteasome inhibitors compared to primary cells (50). Our
data suggest that in primary keratinocytes, activation of the
mitochondrial apoptotic pathway is required for the execution
of TRAIL-induced apoptosis and that proteasome inhibitors
enable the release of proapoptotic molecules such as Smac/
DIABLO, cytochrome c, and HtrA2/Omi from mitochondria
in response to TRAIL treatment.
Our data showing partial cleavage of caspase 3 in primary
keratinocytes are consistent with recent reports demonstrating
that TRAIL-induced activation of Smac/DIABLO release is
dependent on Bax and Bak, promoted by Bid, and inhibited by
Bcl-2 and Bcl-x
L
downstream of caspase 8 activation in differ
-
ent tumor cell lines (12, 69). In line with these reports, the
release of Smac/DIABLO, HtrA2/Omi, and cytochrome c in
the presence of the proteasome inhibitor correlated with full
maturation of caspase 3 to the 17-kDa fragment in primary
keratinocytes. Interestingly, analysis of several Bcl-2 family
members, including Bcl-X
L
, Bax, and Bid, did not reveal
changes in the expression levels or cleavage patterns of these
molecules at time points when full caspase 3 maturation and
release of mitochondrial proapoptotic proteins is already de-
tected in primary keratinocytes (data not shown). Thus, it
remains to be determined if modulation of the expression
levels of Bcl-2 family members and their translocation to mi-
tochondria or other potential targets are modulated by protea-
some inhibitors.
Moreover, it is possible that cytochrome c-mediated activa-
tion of the apoptosome (5, 7) might also be involved in in-
creased caspase 3 activation. However, our data clearly show
that synergistic action of mitochondrion-derived proapoptotic
molecules in concert with death receptor-mediated caspase 3
activation is ultimately necessary and sufficient to overcome
inhibitory molecules such as XIAP in primary keratinocytes. In
support of this concept, recent in vitro experiments demon-
strated that an increase in Smac/DIABLO levels can overcome
XIAP-mediated inhibition of caspase 3 maturation (69). Of
interest, the removal of the prodomain of the effector caspase
6 is required for full enzymatic activity, further supporting our
findings for caspase 3 (10).
When comparing the dose-dependent cleavage patterns of
TRAIL-resistant primary keratinocytes and highly sensitive
transformed keratinocytes, low concentrations of TRAIL were
sufficient to induce fully active p17 fragments in transformed
cells but not in primary cells, indicating that primary keratin-
ocytes require an additional signal for full caspase 3 matura-
tion. As shown in this study, one explanation for this difference
is the differential expression of XIAP, which inhibits the auto-
proteolytic maturation of caspase 3 (14, 15). Primary keratin-
ocytes might require the release of Smac/DIABLO and HtrA2/
Omi (64) from the mitochondria for inactivation of XIAP.
Thus, the stoichiometry of DISC-generated active caspase 8,
cytoplasmic XIAP, and mitochondrion-released Smac/DIA-
BLO or HtrA2/Omi might define resistance or sensitivity to
TRAIL-induced apoptosis in primary keratinocytes and possi-
bly also in other cells, as suggested recently (69).
Remarkably, there may be important cell type-specific dif-
ferences in the action of proteasome inhibitors, as primary
human hepatocytes are not sensitized to TRAIL by these
agents (T. Ganten and H. Walczak, unpublished data). These
differences might be explained by differential expression of
TRAIL receptors, distinct DISC components, or downstream
inhibitory molecules, as described in this report. Our data
demonstrating interaction of XIAP with caspase 3 under phys-
iological conditions as well as increased resistance to TRAIL-
induced apoptosis following ectopic expression of XIAP in
highly TRAIL-sensitive transformed keratinocytes further sup-
port the important role of XIAP in TRAIL resistance in pri-
mary keratinocytes.
Taken together, our data support a model in which pro-
teasome inhibitors allow initiator caspase-mediated activa-
tion of mitochondrial pathways of apoptosis in primary cells.
The activation of mitochondrial pathways is necessary for
inactivation of XIAP and, consequently, of full caspase 3
activity in primary keratinocytes (Fig. 10). Alternatively,
XIAP might exert its caspase-inhibitory role by its ubiquitin-
protein ligase activity (70). Since polyubiquitination labels
proteins for proteasomal degradation, proteasome inhibi-
tion may thereby lead to increased levels of active caspase 3
and subsequent apoptotic cell death in cells expressing high
levels of XIAP. This mechanism might not be required in
cells expressing low levels of XIAP, as supported by our
VOL. 23, 2003 EFFECTOR CASPASE INHIBITION IN PRIMARY KERATINOCYTES 787

overexpression studies. However, another possibility of the
action of the proteasomal inhibitor may be decreased XIAP-
mediated degradation of Smac/DIABLO, as suggested by a
very recent report (47). Future studies are required to de-
lineate these points.
It is possible that the resistance mechanism of primary
human keratinocytes outlined in this study is also relevant
for other types of primary cells and could thus be a more
general mechanism of resistance of primary cells to TRAIL.
In that respect, our findings suggest that the use of protea-
some inhibitors as anticancer agents might lead to unwanted
sensitization of primary cells to death receptor-mediated
apoptosis. Thus, the clinical application of proteasome in-
hibitors, either alone or in combination with death receptor
agonists such as TRAIL, must be closely monitored for
unwanted toxicity that could be due to the apoptosis-sensi-
tizing effect exerted by these agents on primary cells as
identified in this study.
ACKNOWLEDGMENTS
We thank P. H. Krammer for monoclonal antibodies to caspase 8
(C-15) and cFLIP (NF-6), D. Nicholson for caspase 3 and XIAP
antisera as well as biotin-DEVD-aomk, M. MacFarlane for rabbit
serum to Smac/DIABLO, X. Wang for rabbit serum to Bid, R. Taka-
hashi for rabbit serum to HtrA2/Omi, S. Roy for the protocol to
perform affinity labeling experiments, and C. S. Duckett for the XIAP
expression vector. We are also grateful to Evi Horn for excellent
technical assistance and Heiko Stahl for generating monoclonal anti-
bodies to TRAIL-R1 to -R4.
Part of this study was funded by grants from the IZKF Wu¨rzburg
(01 K5 9603), Sander-Stiftung (2000.092.1), and Deutsche Kreb-
shilfe (10-1951-Le1) to Martin Leverkus. Henning Walczak is sup-
ported by a BioFuture grant from the Bundesministerium fu¨r Bil-
dung und Forschung (BMBF). Manfred Neumann and Bernd
Baumann were supported by grants from the Deutsche Forschungs-
gemeinschaft (NE 608/1-3 and/2-2 to M.N. and SFB 497/B1 to
B.B.).
The first two authors contributed equally to this paper.
The last two authors share senior authorship of this paper.
FIG. 10. Model of action of proteasome inhibitors mediating sensitization of primary keratinocytes to TRAIL. TRAIL activates caspases 8 and
10 at the DISC. Initial processing of caspase 3 produces a p20/p12 intermediate of caspase 3 which is inhibited by XIAP. Proteasome inhibitors
interfere with caspase 8-mediated activation of mitochondrial pathways of apoptosis, leading to the caspase 8-activated release of Smac/DIABLO
and HtrA2/Omi and subsequent removal of the caspase 3 prodomain (hatched bars). This activation of mitochondrial pathways is necessary for
inactivation of XIAP and subsequent full caspase 3 activity in primary keratinocytes.
788 LEVERKUS ET AL. M
OL.CELL.BIOL.

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