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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2249-2258
A20 Inhibits NF- B Activation in Endothelial Cells Without
Sensitizing to Tumor Necrosis Factor-Mediated Apoptosis
By
Christiane Ferran,
Deborah M. Stroka,
Anne Z. Badrichani,
Jeffrey
T. Cooper,
Christopher J. Wrighton,
Miguel Soares,
Shane T. Grey, and
Fritz H. Bach
From the Department of Surgery, Center por Immunobiology, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston MA; and
Therexsys Ltd, The Science Park, Keele University, Keele,
Staffordshire, UK.
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ABSTRACT |
Expression of the NF- B-dependent gene A20 in endothelial cells
(EC) inhibits tumor necrosis factor (TNF)-mediated apoptosis in the
presence of cycloheximide and acts upstream of IB degradation to
block activation of NF-B. Although inhibition of NF-B by IB
renders cells susceptible to TNF-induced apoptosis, we show that when
A20 and IB are coexpressed, the effect of A20 predominates in
that EC are rescued from TNF-mediated apoptosis. These findings place
A20 in the category of "protective" genes that are induced in
response to inflammatory stimuli to protect EC from unfettered activation and from undergoing apoptosis even when NF-B is blocked. From a therapeutic perspective, genetic engineering of EC to express an
NF-B inhibitor such as A20 offers the mean of achieving an anti-inflammatory effect without sensitizing the cells to TNF-mediated apoptosis.
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INTRODUCTION |
ENDOTHELIAL CELLS (EC), which are
frequently exposed to the pleiotropic cytokine tumor necrosis factor
(TNF) at sites of inflammation, are usually resistant to TNF-mediated
apoptosis. This resistance is mediated by de novo expression of a set
of "protective genes." Recent reports pointed to the critical
role of the transcription factor NF-B in the induction of those
protective genes.1-3 As recently shown by several groups,
blockade of NF-B activation by overexpression of its inhibitor
IB or by knocking out the p65/RelA sensitizes embryonic
fibroblasts, macrophages, jurkat cells, and a fibrosarcoma cell line to
TNF-induced apoptosis.1-3 We have previously reported a
similar finding in primary EC (C.J. Wrighton et al, personal
communication, September 1995).
We recently showed that the expression of A20, a zinc finger protein
originally identified as a TNF-inducible gene in human umbilical vein
endothelial cells (HUVEC)4 and shown to be dependent on
NF-B for its expression,5,6 inhibits activation of
NF-B in EC.7 Our studies showed that the expression of
A20 in EC suppressed the activation of a reporter that is dependent
solely on NF-B as well as reporters for several of the
NF-B-dependent genes, including E-selectin, interleukin (IL)-8,
IB, and tissue factor, that are upregulated when EC are
activated.8-12
In the present studies, we transduced primary EC with a recombinant A20
adenovirus (rAd.A20), which leads to high levels of A20 protein
expression in almost 100% of cells. This allowed us (1) to confirm
that expression of A20 would inhibit upregulation of NF-B-dependent
genes in their normal DNA context, (2) to dissect the level at which
NF-B inhibition occurs, and (3) to evaluate whether inhibition of
NF-B in EC by A20 would sensitize the cells to TNF-mediated
apoptosis, as with IB, or whether the antiapoptotic function of
A20 would prevent such sensitization. Although A20 was described based
on its antiapoptotic function in B cells and fibroblasts,13,14 this property has not been tested in EC.
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MATERIALS AND METHODS |
Adenoviral-mediated gene transfer to porcine aortic endothelial cells.
Fresh EC were isolated from porcine aortas15 by scraping
and cultured in Dulbecco's modified Eagle's Medium (DMEM)
supplemented with 10% heat-inactivated fetal calf serum (FCS; High
Clone, GIBCO-BRL, Grand Island, NY) and 50 U/mL
penicillin/streptomycin. Ninety percent to 100% of confluent porcine
aortic endothelial cells (PAEC) monolayers from the fifth or the sixth
passage were infected with the rAd.A20, the rAd.IB, or the
control rAd. -gal at a moiety of infection (MOI) of 500 in 1% FCS
DMEM supplemented with penicillin (125 U/mL), streptomycin (125 mg/mL),
and L-glutamine (2 mmol/L; all purchased from GIBCO-BRL) and incubated
for 1.5 hours in a 5% CO2 humid incubator on a rocking
platform. Following the initial 1.5 hours, FCS-enriched medium was
added to the rAd-infected cells to achieve a 10% FCS final
concentration. Twenty-four hours following the infection, the medium
was changed and the cells allowed to rest for an additional 24 hours
before being assessed for the expression and the function of the
transferred gene. Infection of PAEC with 2 rAd. (rAd.IB and
rAd.-gal or rAd.A20) was achieved at a combined MOI of 1,000. Expression of the transgenes was evaluated by immunohistochemistry
labeling using a mouse anti-human A20 monoclonal antibody (MoAb) (kind
gift of Dr Vishva Dixit, University of Michigan, Ann Harbor), a rabbit
anti-IB polyclonal (MAD-3) anti-serum (Santa-Cruz Biotechnology,
Santa Cruz, CA) that cross-reacts with the porcine IB
protein,16 or Northern blot analysis using specific
radiolabeled cDNA probes.
Recombinant adenoviruses.
The rAd.A20 is a kind gift of Dr. Vishva Dixit; the rAd.-gal, used
as a control adenovirus, is a kind gift of Dr Robert Gerard (University
of Texas SW); and the rAd.IB was generated by C.J. Wrighton as
described and expresses the porcine IB gene
(ECI-6).16 In brief, construction of these rAd. was done by
cloning the respective gene's cDNA in the
pAC.CMV-pLpASR+ vector as
described.17 This A20 pAC plasmid was then cotransfected with pJM17, a recombination plasmid system developed by McGrory et
al18 in the 293 embryonic kidney cell line. To reduce the possibility of wild-type virus being produced from a plasmid that contains adenovirus genomic DNA, the pJM17 vector had a plasmid vector
sequence inserted in the E1 region, which makes the DNA molecule too
large to package in an adenovirus particle.19 Production of
rAd. was done in the embryonic kidney 293 cell line. Recombinant adenoviruses were subsequently purified by two consecutive cesium chloride centrifugations and tittered by limiting dilution on 293 cells.
Reagents.
PAEC were stimulated with either 100 ng/mL of lipopolysaccharide (LPS;
Escherichia coli 0B55; Sigma, St Louis, MO), 100 U/mL of
recombinant human TNF (kind gift of Novartis
Pharmaceuticals, East Hanover, NJ), 5 × 10 8 mol/L of phorbol 12-myristate 13-acetate (PMA), 300 µmol/L of hydrogen peroxide (H2O2), or 10 U/mL of human -thrombin (Sigma). In some experiments the inhibitor
of serine proteases, dichloroisocoumarin (Sigma), was added 30 minutes
before the given agonist at the concentration of 25 µmol/L. The
inhibitor of translation cycloheximide (CHX) used at a final
concentration of 2 µg/mL and the propidium iodide (PI) used in
apoptosis assays were purchased from Sigma.
Nuclear extracts and electrophoretic mobility shift assay (EMSA).
Nuclear proteins were extracted from PAEC before stimulation and 2 hours after stimulation with TNF according to the method described
elsewhere.20 Protease inhibitors were added in all buffers
used during nuclear extraction, namely phenylmethyl sulfonyl fluoride
(PMSF); 50 µg/mL), leupeptin (0.5 µg/mL), antipain (0.5 µg/mL),
aprotinin (0.5 µg/mL), pepstatin (1 µg/mL), benzamidine (100 µg/mL), chymostatin (100 µg/mL), TLCK (50 µmol/L),
and TPCK (100 µmol/L). Protein concentration of nuclear extracts was
determined by the Bradford assay.21 Bovine serum albumin
was used as the standard. Nuclear extracts were frozen on dry ice and
stored at  80°C until assessed in EMSA. The probes used in EMSA
were labeled by random priming with -[32P]-dATP (80 µCi at 3,000 Ci/mmol; Amersham, Arlington Heights, IL) using the
Klenow fragment of E coli DNA polymerase I in the presence of
nonlabeled dTTP, dCTP, and dGTP. Binding reactions in 25 µL contained
100,000 cpm of double-stranded oligonucleotide; 3 µg of poly (dI-dC);
and 5 µg of nuclear extract proteins in 20 mmol/L HEPES pH 7.9, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L -mercaptoethanol, and 5%
glycerol; they were performed for 30 minutes at room temperature (RT).
For electrophoresis, high ionic strength 6%-polyacrylamide gels were
used as described.22 A double-stranded oligonucleotide
containing the second porcine ECI-6 (IB) oligonucleotide
(BS-2-5'-AATTCGGCTTGGAAATTCCCCGAGCG-3') was
used for NF-B EMSA.23
Cytoplasmic extracts and Western blot analysis of
IB expression.
Cytoplasmic extracts were prepared before, 10 minutes after, and 2 hours after TNF (T) treatment from noninfected (NI), rAd.A20, or
rAd.-gal-infected PAEC, as described.16 Protein
concentration of these cytoplasmic extracts was quantitated by the
Bradford method. Twenty micrograms of protein per sample was evaluated by Western blot analysis for IB expression, as
described.16 IB was detected using anti-MAD-3 rabbit
polyclonal IgG antiserum (#C-21) from Santa-Cruz Biotechnology and a
peroxidase-conjugated donkey anti-rabbit secondary antibody (Pierce,
Rockford, IL) followed by enhanced chemiluminescence (ECL) detection
(Amersham).
RNA extraction and Northern blot analysis.
RNA was extracted from PAEC expressing or not the different transgenes
before and 2 hours following stimulation by the different agonists.
Total RNA was isolated using Trizol reagent (GIBCO-BRL) according to
the method described by Chomczynski and Sacchi.24 The
amount of extracted RNA was calculated from optical density (OD)
measurements at 260 nm (1 OD260 ~ 40 µg/mL). Purity
and integrity of RNA samples were confirmed in 1% agarose gels
containing ethidium bromide. For Northern blot analysis, equal amounts
of RNA (10 µg per lane) were loaded and run on a 1.3%
agarose/formaldehyde gel. RNA was then transferred to a nylon membrane
and hybridized to cDNA probes encoding for porcine
E-selectin25; porcine IL-8 derived by E. Hofer (unpublished
observation, September 1993); porcine IB
(ECI-6)23; human vascular cell adhesion molecule (VCAM)-1
that was shown to cross-hybridize with its porcine homologue, a kind
gift of Dr T. Collins (Children's Hospital, Boston MA); junB, a kind
gift of Dr Ulrich Rulter (Heidelberg, Germany); and an A20 cDNA probe corresponding to a 250-bp HindIII-released fragment from the
N-terminus of the sequence.4 In all experiments, a cDNA
probe for human glyceraldehyde triphosphate dihydrogenase (GAPDH) was
used to confirm equal loading of RNA in all the wells. All probes were labeled with -[32P]-dATP (Amersham) using a random
primer labeling kit (Stratagene, La Jolla, CA).
E-selectin enzyme-linked immunosorbent assay (ELISA).
Noninfected or rAd.-infected PAEC were grown to confluence in 96-well
microtiter plates. These cells were then stimulated with the different
stimuli prementioned. Four hours following stimulation, the cells were
washed three times with phosphate-buffered saline (PBS), fixed in
ice-cold 0.02% glutaraldehyde at 4°C for 5 minutes. Cells were then
incubated with a mouse MoAb (BBA1) directed against human
E-selectin26 and shown to cross-react with porcine
E-selectin. The BBA1 MoAb was purchased from R&D Systems (Minneapolis,
MN) and used at a 1:10,000 dilution of the hybridoma supernatant. A
goat anti-mouse peroxidase-coupled polyclonal antibody purchased from
Pierce was used as secondary antibody. Optical density was determined
at 490 nm on an LKB ELISA reader.
Cell viability assay.
Cell viability was assessed by means of the vital dye crystal violet
uptake. In brief, cells are stained for 5 minutes with crystal violet
solution then washed thoroughly under tap water. Colored cell
monolayers are then dried and the crystals subsequently dissolved in
10% acetic acid before read on an LKB ELISA reader at 405 nm
wavelength. OD values of NI, nontreated cells were considered to
reflect 100% cell viability.
Apoptosis assay.
Cell death by apoptosis was assessed by flow cytometric analysis of DNA
content. Briefly, following treatment PAEC were obtained, washed twice
with PBS, resuspended in ice-cold 70% ethanol with gentle vortexing to
a final concentration of 1 × 106 cells/mL, and stored at
4°C until analysis. Before quantification of DNA content, PAEC were
pelleted at 400g for 5 minutes, resuspended into PBS, pelleted
again, and resuspended into 200 µL PBS, 0.1% Triton X, 0.1 mmol/L
EDTA, 50 µg/mL DNAse free RNAse (Stratagene) with 5 µg/mL PI. DNA
content analysis was conducted using a FACScan bench top model (Becton
Dickinson, San Jose, CA) with Cellquest acquisition and analysis
software (Becton Dickinson). Cellular debris and doublets were excluded
from analysis by their forward-light-scatter and
right-angle-light-scatter properties.
| |
RESULTS |
Adenoviral-mediated gene transfer of A20 achieves 100% expression in
primary EC cultures.
Primary PAEC were infected with the rAd.A20 at an MOI of 500/cell,
previously shown to be optimal for achieving high levels of expression
in PAEC without causing significant cytotoxicity.16 Immunohistochemical staining of EC transduced with the rAd.A20 using a
mouse anti-human A20 MoAb shows high level of A20 expression in almost
all PAEC 24 to 48 hours following infection (Fig
1). As shown by the labeling, the
expression of A20 is mainly cytoplasmic, which is comparable to its
endogenous expression.14 The anti-A20 MoAb used does not
cross-react with the porcine A20.
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| Fig 1.
Adenoviral-mediated gene transfer of A20 achieves high
levels of expression in cultured PAEC. Cultured 90% to 100% confluent PAEC, from the fifth or the sixth passage, were either not infected (untreated) or infected with either the control rAd.-gal
(adeno-beta-gal) or rAd.A20 (adeno A20). Forty-eight hours following
transduction, PAEC were recovered and cytospinned on glass slides using
a cytospin 3 apparatus (Shandon Inc, Pittsburgh, PA) before being
assessed by immunohistochemistry for the expression of the A20
transgene. Immunohistochemical analysis was performed using a mouse
anti-human A20 MoAb of the IgG1 isotype, followed by a secondary
peroxidase-conjugated goat anti-mouse antibody (Dako, Carpinteria, CA;
right panel). A nonrelevant mouse MoAb was used as a
control (left panel). Results show low to undetectable levels of A20 in
noninfected or rAd.-gal-infected PAEC. In contrast PAEC transduced
with the rAd.A20 showed high levels of expression of A20 in
>95% of the cells. Expression of the A20 protein was limited to
the cytoplasm.
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A20 expression inhibits NF-B activation upstream of
IB degradation.
Our previous reporter studies showed that expression of A20 inhibited
activation of NF-B. To determine the level at which inhibition takes
place, nuclear as well as cytoplasmic extracts were recovered before,
10 minutes after, and 2 hours after TNF treatment. These extracts were
evaluated by EMSA for NF-B binding and by Western blot analysis for
IB expression. The data show that overexpression of A20 in EC
inhibits translocation of NF-B to the nucleus following TNF
treatment by stabilizing IB, ie, inhibition is at a level
upstream of IB degradation (Fig 2A and
B). EMSA analysis of nuclear extracts from
PAEC expressing A20 showed almost no inducible binding for NF-B
consensus DNA sequences as opposed to the high binding detected in
noninfected or rAd.-gal-infected PAEC 2 hours following TNF
stimulation. The moderate binding activity for NF-B in the
A20-expressing cells reflects the presence of a small number of
nontransduced PAEC contaminating the cells expressing A20. Specificity
of DNA binding in both the noninfected and the A20-expressing PAEC was tested by the use of excess cold wild-type (wt) or mutant (mutB) B probes (Fig 2A). Furthermore, we show that A20 expression in PAEC
inhibits the usual IB degradation that occurs 10 minutes following TNF stimulation (Fig 2B), pointing to the fact that A20
expression inhibits NF-B activation by stabilizing IB
expression. A second faster-migrating band is reproducibly detected at
2 hours in the A20-expressing EC. This second band most probably
relates to a degradation product of IB and disappears if PAEC are
pretreated (30 minutes) with low levels (25 µmol/L) of the protease
inhibitor dichloroisocoumarin that is not sufficient on its own to
inhibit IB degradation following TNF treatment (data not shown).
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| Fig 2.
(A) Inhibition of NF-B nuclear DNA-binding activity
following TNF stimulation in rAd.A20-infected PAEC. Nuclear extracts were prepared from noninfected (NI), rAd.A20-infected, or
rAd.-gal-infected PAEC before and 2 hours following treatment with
TNF(T) (100 U/mL) as described in Materials and Methods. NF-B
activation and binding to a specific B binding oligo derived from
the porcine IB promoter was evaluated by means of EMSA as
described. Results reveal that nuclear extracts from PAEC expressing
A20 had almost no inducible binding activity for NF-B binding sites.
Specificity of DNA binding was tested by the use of excess cold
wild-type as a specific competitor (wt) or a mutant B probe
(mutB) used as a nonspecific competitor. Results shown are
representative of three independent experiments. (B) Western blot
analysis of IB expression following TNF treatment. Cytoplasmic
extracts from NI, rAd.A20, and rAd.-gal-infected PAEC were
recovered before and 10 minutes and 2 hours following TNF treatment and
assessed for IB expression by means of Western blot analysis.
Results show that A20 expression in PAEC inhibits the usual IB
degradation that occurs 10 minutes following TNF stimulation. A second
faster-migrating band is detected at 2 hours in the A20-expressing EC.
Results shown are representative of three independent experiments.
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A20 expression in EC inhibits the upregulation of
NF-B-dependent genes in an agonist-independent manner.
The inhibition of NF-B is reflected by the decreased induction of
several NF-B-dependent genes in the presence of A20. Results show
that A20 expression in PAEC (confirmed by Northern analysis for A20
mRNA) significantly inhibits E-selectin, VCAM-1, and IB gene
induction (>80% to 90%) following stimulation by TNF, LPS, and PMA
as compared with high levels of induction in either NI cells or
rAd.-gal-infected cells. A20 expression also achieved significant
inhibition (>70%) of IL-8 gene induction. This effect of A20 is
agonist independent; inhibition was seen when EC were stimulated with
TNF, LPS, or PMA (Fig 3A). This inhibitory
effect on NF-B-dependent genes was confirmed at the protein level
for E-selectin and extended to two other stimuli including human
-thrombin (Th) and H2O2 (H) (Fig 3B). These
findings do not support the suggestion that the inhibitory effect of
A20 is limited to TNF and IL-1 signaling.27 The
induction of a non-NF-B-dependent proto-oncogene
junB28-30 was not decreased by A20 expression as measured
at the mRNA level (Fig 4). The
proto-oncogene junB remained inducible in rAd.A20-infected PAEC
following TNF and LPS stimulation. The level of induction was even
greater than that seen in the rAd.-gal-infected PAEC, although this
difference was not significant when corrected with the expression of
the house keeping gene GAPDH.
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| Fig 3.
(A) Northern blot analysis of E-selectin, VCAM-1, IL-8,
and IB gene induction. PAEC were noninfected (NI), rAd.A20, and rAd.-gal-infected at a MOI of 500 as in Fig 1. Forty-eight hours following infection, PAEC were either left nontreated (C) or were stimulated with TNF (T) (100 U/mL), LPS (L) (100 ng/mL), or PMA (P)
(5.108 mmol/L). A20, E-selectin, VCAM-1, IL-8, IB,
and GAPDH steady-state transcript levels were quantitated in these
samples by Northern blot analysis using
-[32P]-dATP-labeled homologous or cross-reactive
cDNA probes as described in Materials and Methods. Results show that
A20 expression in PAEC (confirmed by Northern analysis for A20 mRNA)
significantly inhibits E-selectin, VCAM-1, and IB gene induction
(>80% to 90%) following stimulation by TNF, LPS, and PMA as
compared with high level of induction in either NI cells or
rAd.-gal-infected cells. A20 expression also achieved significant
inhibition (>70%) of IL-8 gene induction. Results shown are
representative of three independent experiments. (B) Inhibition of
cell-surface expression of the EC-specific adhesion molecule
E-selectin. Confluent PAEC in 96-well microtiter plates were infected
as in (A). Triplicate wells of PAEC were either untreated (C) or
treated with the same stimuli as in (A) extended to -thrombin (Th)
and H2O2 (H) (300 µmol/L). Expression of the
E-selectin protein was analyzed by ELISA 4 hours following stimulation.
Results confirm and extend to -thrombin and oxidative stimuli, the
previous mRNA results, by showing that A20 abrogates surface-expression
of E-selectin in PAEC for all stimuli tested. Results shown are
representative of three independent experiments.
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| Fig 4.
Induction of the non-NF-B-dependent gene junB is not
inhibited by expression of A20. PAEC were infected and stimulated as in
(A) with TNF (T) and LPS (L), and RNA was extracted. Steady-state mRNA
levels of A20, junB, and GAPDH were evaluated by Northern blot analysis
as described in (A) using a junB cDNA probe shown to cross-react with
its porcine homologue. Results show that the proto-oncogene junB is
inducible in rAd.A20-infected PAEC, following TNF and LPS stimulation.
The level of induction was even greater than that seen in the
rAd.-gal-infected PAEC, although this difference was not
significant when corrected for GAPDH.
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A20 expression rescues cycloheximide-sensitized EC from TNF-mediated
apoptosis.
In addition to the suppressive effects just discussed, expression of
A20 in PAEC protected those cells against TNF-mediated cell death in
the presence of an inhibitor of protein translation, ie, CHX. In the
absence of A20, TNF induces apoptosis in CHX presensitized PAEC. Using
crystal violet uptake as an indicator of cell viability, no viable
cells were seen in NI or rAd.-gal-infected PAEC 7 hours after
treatment with CHX/TNF as opposed to more than 60% to 70% viable
cells in rAd.A20-infected PAEC treated or not with CHX (2 µg/mL) 30 minutes before the addition of TNF (100 U/mL; Fig 5A). To confirm that cell
death occurred by apoptosis, DNA fragmentation was determined by PI
labeling followed by flow cytometric measurement of the percentage of
nuclei with hypodiploid DNA content, as previously described.31 This method allows defining four regions
within a flow cytometric cell cycle histogram: a major diploid peak
(G0/G1), a small hyperdiploid region (S), and
a minor tetraploid peak (G2/M). Cells in the region below
the G0/G1 peak, designated A0, are
cells undergoing apoptosis-associated DNA fragmentation. In noninfected quiescent PAEC cultures the percentage of cells in the A0
region varied between 1% and 7%. This percentage was not modified,
when PAEC were transduced with rAd.A20 or rAd.-gal (assessed 48 hours following infection; Fig 5B, left panel). Similarly, this
percentage was not modified in NI, rAd.A20, and rAd.-gal-infected
PAEC when treated with CHX or TNF alone (data not shown). Upon
treatment with CHX and TNF, the percentage of apoptotic cells increased to 30% to 40% in the NI or rAd.-gal-infected PAEC, whereas it remained comparable to control cells in A20-expressing PAEC (Fig 5B,
right panel). These later results paralleled those obtained with the
crystal violet uptake, thus validating its use for further experiments.
Taken all together, our results show that A20 serves two functions in
EC: its expression downregulates EC activation through the inhibition
of NF-B and protects from TNF-induced programmed cell death.
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| Fig 5.
(A) Overexpression of A20 rescues
CHX-sensitized EC from TNF-mediated apoptosis. Noninfected, rAd.A20-,
and rAd.-gal-infected confluent monolayers of PAEC were treated 48 hours following infection with 100 U/mL of TNF in the presence or
absence of 2 µg/mL of CHX. Seven hours following treatment, cell
viability was assessed using a vital dye (crystal violet) uptake assay
as described. Results are expressed as percentage of survival compared
with NI, nontreated (control) PAEC whose values were considered to represent 100% of cell survival. Results shown are the mean ± SEM of
triplicate wells and are representative of three independent experiments. A20 expression significantly protects PAEC from
CHX/TNF-induced cytotoxicity. No viable cells were seen in NI or
rAd-gal-infected PAEC treated with CHX/TNF, as opposed to more than
60% to 70% viability in rAd.A20-infected PAEC treated or not with CHX
(2 µg/mL) 30 minutes before the addition of TNF (100 U/mL). (B)
Overexpression of A20 prevents apoptotic fragmentation of cellular DNA
in CHX- and TNF-treated PAEC. Noninfected PAEC or PAEC infected with
either rAd.-gal or rAd.A20 were treated with CHX (2 µg/mL) or TNF
(100 U/mL) either alone or in combination for 7 to 8 hours. Cells were then obtained and assessed for apoptosis-induced DNA fragmentation as
described in Materials and Methods. The region below the
G1/G0 peak, designated A0,
represents cells undergoing apoptosis with fractional DNA content and
is presented as a percentage of the total events collected. Results
obtained correlated with the crystal violet uptake data, validating its
use for further experiments.
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Expression of A20 in rAd.IB-transduced EC overcomes
sensitization to TNF-mediated apoptosis.
Overexpression of IB in PAEC sensitizes them to TNF-mediated
apoptosis as confirmed using the flow cytometric analysis of DNA
content as well as crystal violet uptake (Fig 6A and
C). Having established that A20 prevents
TNF-mediated apoptosis in EC even though it inhibits activation of
NF-B, we tested whether sensitization to TNF-induced apoptosis when
NF-B is inhibited by I B would be overcome by the expression
of A20. PAEC were cotransduced with two rAd. (rAd.IB and rAd.A20
or rAd.-gal) at a combined MOI of 1,000 that achieves significant
expression of both transgenes in cultured PAEC as confirmed by Northern
blot analysis testing for mRNA expression of both the IB and the
A20 transgenes (Fig 6B). At this MOI, cytotoxicity remained below 10%
of the cultured cells when assessed by an LDH enzyme release assay,
used as an indicator of membrane rupture. Released enzyme was assayed
using a commercially available test system (CytoTox 9600 non-radioactive cytotoxicity kit; Promega, Madison, WI) and the data
obtained evaluated according to the manufacturer's instructions (data
not shown). At this MOI, PAEC cotransduced with both the rAd.IB and the rAd.A20 were protected from apoptosis when stimulated with TNF
(Fig 6C). In contrast, PAEC expressing IB alone underwent apoptosis under the same conditions. Results show that coexpression of
A20 in rAd.IB-infected PAEC results in a significant increase in
cell viability following TNF treatment (60% ± 2) as opposed to
cells cotransduced with the rAd.-gal, where only 19% of the cells
were still viable (a percentage that is not significantly different
from the cells transduced with the sole rAd.IB [28% of viable
cells]; Fig 6C). The effect of A20 is dominant; the EC are rescued
from sensitization to TNF-mediated apoptosis by IB. These results
are in contrast with those of Beg and Baltimore1 in which
A20 expression was not able to rescue
RelA/ negative fibroblasts from
TNF-mediated cytotoxicity. We suggest that one possible reason for the
difference in our findings may be that we performed our studies in EC,
whereas the other studies quoted used other cell types.
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| Fig 6.
(A) Expression of IB in PAEC sensitizes them to
TNF-mediated apoptosis. Noninfected or rAd.IB-infected PAEC were
treated for 7 hours with TNF (100 U/mL). Cells were then obtained as
described and assessed for apoptosis-induced DNA fragmentation. TNF
treatment did not affect the percentage of cells in the A0
region, whereas this percentage was increased to 42% in
IB-expressing PAEC. (B) Infection of cultured PAEC with rAd.A20
and rAd.IB results in the coexpression of both transgenes as
assessed by Northern blot analysis. (C) Coexpression of A20 in
rAd.IB-infected EC reverts their phenotype to resistance against
TNF-mediated apoptosis. PAEC were cotransduced with the rAD.IB at
MOI of 500 for each virus. Noninfected PAEC or PAEC infected with the
rAd.IB (500 MOI) alone or in combination with the rAd.A20 (500 MOI) or rAd.-gal (500 MOI) were treated with TNF for 7 hours, after
which time cell viability was assessed by crystal violet uptake.
Results show that coexpression of A20 in rAd.IB-infected PAEC
results in a significant increase in cell viability following TNF
treatment (60% ± 2) as opposed to cells cotransduced with the
rAd.-gal, where only 19% of the cells were still viable (a
percentage that is not significantly different from the cells
transduced with the sole rAd.IB [28% of viable cells]).
Results are also expressed as percentage of survival compared with the
noninfected, nontreated (control) PAEC whose values were considered to
represent 100% of cell survival. Results shown are representative of
three independent experiments.
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DISCUSSION |
In this study, we have established that the A20 gene can classify
within the category of cytoprotective genes in the endothelium, ie,
genes that are upregulated in response to inflammatory stimuli such as
TNF and act to protect EC from apoptosis and to limit the damage
associated with activation.32 Indirect in vivo evidence for
such an effect is suggested by our studies in a hamster heart to rat
xenotransplantation model.33 We have shown that in grafts that achieve long-term survival, EC express A20. This expression is
correlated with the absence of signs of activation or apoptosis. In
contrast, EC in rejected grafts do not express A20 and show evidence of
activation and apoptosis. Presumably A20 has the same functions in vivo
as in vitro: suppression of EC activation and protection from
apoptosis.
We confirm that adenoviral-mediated overexpression of A20 in EC acts as
a potent inhibitor of EC activation by inhibiting at the
transcriptional level the upregulation of several genes (expressed
within their DNA context) implicated in the acquisition of the EC of a
proinflammatory phenotype.34 We further show that the
inhibitory effect of A20 upon EC activation is related to the blockade
of the transcription factor NF-B at a level upstream of IB
degradation. This effect is so far specific; the expression of A20 in
the EC did not have any effect on the SP-1 or cyclic adenosine
monophosphate response element (CRE) transcription factors. Indeed, A20
expression did not affect the induction by the viral protein c-tat of a
human immunodeficiency virus (HIV) reporter that depends on SP-1 for
its induction.7 In addition, the expression of A20 did not
modify the binding of the transcription factor CRE in EMSA (data not
shown) or affect the upregulation of the immediate-early response gene
junB, a member of the AP-1 transcription factors that is
transcriptionally regulated by CRE-like and STAT family
proteins.28,30 These results do not preclude a potential effect of A20 on other transcription factors. Indeed, a report in the
literature shows that expression of A20 inhibits the induction of an
AP-1-dependent reporter in a breast tumor cell line.27 However, if such an inhibition occurs also in EC, it would need to
happen in a manner that does not alter AP-1 binding in EMSA. Nuclear
extracts from rAd.A20 and rAd.-gal-infected PAEC before and after
TNF stimulation showed similar binding to a consensus AP-1 radiolabeled
oligomer (data not shown). The effect of A20 in EC upon AP-1
transactivating properties still needs further analysis. A different
effect on AP-1-mediated transcription in EC, as opposed to the breast
tumor cell line, would not be surprising. Differences in the function
of A20 according to the cell type have been reported, ie, A20
overexpression protects B cells but not the breast carcinoma cell line
MCF7S1 against serum starvation-mediated apoptosis.14,27
The precise mechanism by which A20 inhibits the signaling pathway
leading to NF-B activation is yet to be determined. The inhibitory
effect of A20 being localized to the Zn-finger domains of the A20
molecule35 (and manuscript in preparation),
which can bind high levels of the known antioxidant element Zn, might suggest an antioxidant mechanism.36,37 Antioxidants, such
as pyrrolidine dithiocarbamate (PDTC), are potent inhibitors of NF-B activation, and like A20 act at a level upstream of IB
phosphorylation and degradation.22,38 Alternatively, A20
could interact through its Zn-binding domains39,40 with a
molecule(s) critically implicated in the signaling that leads to
NF-B activation. A recent report showed that A20 interacts through
its N-terminus domain with the TNF receptor-associated factors (TRAF)-1
and TRAF-2.35 In the model proposed by the authors, A20
interacts in human cells with TRAF-1 that serves as an anchor to
associate it to TRAF-2 and can then interrupt TNF-mediated signaling
and NF-B activation. However, TRAF-2 is only implicated in TNF and
CD40-mediated NF-B activation,35,41,42 and thus likely
does not explain the inhibitory effect to the other agonists used in
our study. We rather favor that the inhibitory effect of A20, as
already suggested, would affect both a TRAF-2-dependent and
TRAF-2-independent pathways.35 To explain the
agonist-independent nature of the inhibitory effect of A20, one has to
hypothesize that its interaction with a key signaling molecule(s)
should occur at a level that is common to all stimuli studied.
Antiapoptotic genes, such as Bcl-2, have been shown to interact with
molecules involved in signaling pathways such as p21Ras,
p23R-Ras, and Raf-1 kinase43-45 to mediate
their antiapoptotic effect. Bcl-2, which has an effect similar to A20
in blocking NF-B activation and preventing apoptosis in EC (A.Z.
Badrichani et al, in press), interacts with Raf-1 kinase
and targets it to the outer mitochondrial membrane. This translocation
potentially brings Raf-1 kinase into proximity with specific substrates
that are relevant in the life-death balance of the
cell.46-48 In support of this hypothesis, Vincenz et
al49 have recently shown using the yeast two-hybrid system, that A20 associates with the 14-3-3 proteins in an isoform-specific manner. These 14-3-3 proteins function as chaperone and adapter molecules bridging A20 with other molecules, namely, the signaling molecule c-Raf that coimmunoprecipitates with A20 in a
14-3-3-dependent manner.49
In addition, we were able to show that expression of A20 in the EC
protects them against TNF-mediated apoptosis, a function that had not
yet been clearly shown in those cells. This result is of importance as
it shows that effective inhibition of NF-B could be achieved, at
least in EC, without sensitizing these cells to TNF-mediated apoptosis.
The dominant effect of A20 over IB expression paralleled results
achieved with antioxidants, which when added to IB-expressing EC
prevent them from undergoing TNF-mediated apoptosis (C.J. Wrighton et
al, personal communication, September 1995), further
pointing to potential similarities between A20 and antioxidants.
From a therapeutic point of view, blockade of NF-B has been
suggested as a possible approach to two types of problems that represent opposite sides of the same coin. First, inhibition of NF-B
in EC could be used to prevent the proinflammatory consequences of EC
activation, which have been implicated in several pathologic conditions
including allograft and xenograft rejection.34,50,51 To
achieve this goal, a method to block NF-B is needed that would not
sensitize the cells to TNF-induced apoptosis and even protect them
against it. Indeed, EC loss would expose the subendothelial matrix,
which would be equally as detrimental as the consequences of EC
activation itself. Expression of a gene such as A20 that inhibits
inflammatory reactions and still protects the cells from death may
achieve this purpose. We suggest that any such NF-B inhibitory agent
may have to act upstream of IB phosphorylation and
degradation52-55 and inhibit the effects of reactive oxygen species and the activation of proteases and caspases that are prerequisites for activation of NF-B and are part of the molecular machinery leading to apoptosis.52,56-58 Second, inhibition
of NF-B has been suggested as one approach to using TNF for tumor therapy by rendering the cells sensitive to TNF-mediated
apoptosis.1 Our data serve to qualify these suggestions. To
achieve this goal it would be critical to use an inhibitor such as
IB that acts directly and solely on NF-B to prevent
activation. Therapeutic agents such as antioxidants, which were
proposed as one possible agent to sensitize tumor cells to TNF-mediated
apoptosis, or A20 would not be suitable. Although antioxidants and A20
inhibit NF-B, their antiapoptotic properties are dominant over
sensitization to TNF-induced apoptosis, and thus the very effect one
would like to achieve with TNF would not be attained.
| |
FOOTNOTES |
Submitted November 11, 1997;
accepted January 2, 1998.
Supported by a grant from Novartis Pharmaceuticals, Basel, Switzerland.
F.H.B. is a paid consultant to Novartis Pharmaceuticals.
Address reprint requests to Christiane Ferran, MD, PhD, SCI, Beth
Israel Deaconess Medical Center, 99 Brookline Ave, Boston, MA 02215.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank E. Csizmadia for preparation of primary PAEC; Dr Robert Gerard
for the rAd.-gal control adenovirus and for the pAC.CMV transfer
vector; Dr V. Dixit for providing the rAd.A20 and the anti-A20 MoAb;
Drs E. Hofer, R. de Martin, T. Collins, and U. Rulter for providing the
IL-8, IB, VCAM-1, and junB probes; Dr W.W. Hancock for helping in
the immunohistochemistry experiments; and Dr J. Anrather for helpful
discussions.
| |
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M. A. Arruda, A. G. Rossi, M. S. de Freitas, C. Barja-Fidalgo, and A. V. Graca-Souza
Heme Inhibits Human Neutrophil Apoptosis: Involvement of Phosphoinositide 3-Kinase, MAPK, and NF-{kappa}B
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[Abstract]
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C. R. Longo, M. B. Arvelo, V. I. Patel, S. Daniel, J. Mahiou, S. T. Grey, and C. Ferran
A20 Protects From CD40-CD40 Ligand-Mediated Endothelial Cell Activation and Apoptosis
Circulation,
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[Abstract]
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S. T. Grey, C. Longo, T. Shukri, V. I. Patel, E. Csizmadia, S. Daniel, M. B. Arvelo, V. Tchipashvili, and C. Ferran
Genetic Engineering of a Suboptimal Islet Graft with A20 Preserves {beta} Cell Mass and Function
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C. W. Xiao, X. Yan, Y. Li, S. A. G. Reddy, and B. K. Tsang
Resistance of Human Ovarian Cancer Cells to Tumor Necrosis Factor {alpha} Is a Consequence of Nuclear Factor {kappa}B-Mediated Induction of Fas-Associated Death Domain-Like Interleukin-1{beta}-Converting Enzyme-Like Inhibitory Protein
Endocrinology,
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R. Riachy, B. Vandewalle, J. Kerr Conte, E. Moerman, P. Sacchetti, B. Lukowiak, V. Gmyr, T. Bouckenooghe, M. Dubois, and F. Pattou
1,25-Dihydroxyvitamin D3 Protects RINm5F and Human Islet Cells against Cytokine-Induced Apoptosis: Implication of the Antiapoptotic Protein A20
Endocrinology,
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W.-S. Wu, Z.-X. Xu, and K.-S. Chang
The Promyelocytic Leukemia Protein Represses A20-mediated Transcription
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D. R. Laybutt, H. Kaneto, W. Hasenkamp, S. Grey, J.-C. Jonas, D. C. Sgroi, A. Groff, C. Ferran, S. Bonner-Weir, A. Sharma, et al.
Increased Expression of Antioxidant and Antiapoptotic Genes in Islets That May Contribute to {beta}-Cell Survival During Chronic Hyperglycemia
Diabetes,
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[Abstract]
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J. G. Kupfner, J. J. Arcaroli, H.-K. Yum, S. G. Nadler, K.-Y. Yang, and E. Abraham
Role of NF-{kappa}B in Endotoxemia-Induced Alterations of Lung Neutrophil Apoptosis
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A. Tedgui and Z. Mallat
Anti-Inflammatory Mechanisms in the Vascular Wall
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C. W. Xiao, K. Ash, and B. K. Tsang
Nuclear Factor-{{kappa}}B-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor {{alpha}}-Induced Apoptosis
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SUSAN. L. DeMEESTER, T. G. BUCHMAN, and J. P. COBB
The heat shock paradox: does NF-{kappa}B determine cell fate?
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S. Brouard, L. E. Otterbein, J. Anrather, E. Tobiasch, F. H. Bach, A. M.K. Choi, and M. P. Soares
Carbon Monoxide Generated by Heme Oxygenase 1 Suppresses Endothelial Cell Apoptosis
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K. Ikeda, H. Wakimoto, T. Ichikawa, S. Jhung, F. H. Hochberg, D. N. Louis, and E. A. Chiocca
Complement Depletion Facilitates the Infection of Multiple Brain Tumors by an Intravascular, Replication-Conditional Herpes Simplex Virus Mutant
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Y. Lin, M. P. Soares, K. Sato, E. Csizmadia, S. C. Robson, N. Smith, and F. H. Bach
Long-Term Survival of Hamster Hearts in Presensitized Rats
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S. T. Grey, M. B. Arvelo, W. Hasenkamp, F. H. Bach, and C. Ferran
A20 Inhibits Cytokine-Induced Apoptosis and Nuclear Factor {kappa}B-Dependent Gene Activation in Islets
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Y. Lin, M. P. Soares, K. Sato, K. Takigami, E. Csizmadia, N. Smith, and F. H. Bach
Accommodated Xenografts Survive in the Presence of Anti-Donor Antibodies and Complement That Precipitate Rejection of Naive Xenografts
J. Immunol.,
September 1, 1999;
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K. Heyninck, D. De Valck, W. V. Berghe, W. Van Criekinge, R. Contreras, W. Fiers, G. Haegeman, and R. Beyaert
The Zinc Finger Protein A20 Inhibits TNF-induced NF-{kappa}B-dependent Gene Expression by Interfering with an RIP- or TRAF2-mediated Transactivation Signal and Directly Binds to a Novel NF-{kappa}B-inhibiting Protein ABIN
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D. M. Stroka, A. Z. Badrichani, F. H. Bach, and C. Ferran
Overexpression of A1, an NF-kappa B-Inducible Anti-Apoptotic Bcl Gene, Inhibits Endothelial Cell Activation
Blood,
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A. Stempien-Otero, A. Karsan, C. J. Cornejo, H. Xiang, T. Eunson, R. S. Morrison, M. Kay, R. Winn, and J. Harlan
Mechanisms of Hypoxia-induced Endothelial Cell Death. ROLE OF p53 IN APOPTOSIS
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M. P. Soares, A. Muniappan, E. Kaczmarek, K. Koziak, C. J. Wrighton, F. Steinhauslin, C. Ferran, H. Winkler, F. H. Bach, and J. Anrather
Adenovirus-Mediated Expression of a Dominant Negative Mutant of p65/RelA Inhibits Proinflammatory Gene Expression in Endothelial Cells Without Sensitizing to Apoptosis
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U. M. Malyankar, M. Scatena, K. L. Suchland, T. J. Yun, E. A. Clark, and C. M. Giachelli
Osteoprotegerin Is an alpha vbeta 3-induced, NF-kappa B-dependent Survival Factor for Endothelial Cells
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A. Denk, M. Goebeler, S. Schmid, I. Berberich, O. Ritz, D. Lindemann, S. Ludwig, and T. Wirth
Activation of NF-kappa B via the Ikappa B Kinase Complex Is Both Essential and Sufficient for Proinflammatory Gene Expression in Primary Endothelial Cells
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Abstract
Introduction
Abstract