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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3803-3810
Overexpression of A1, an NF- B-Inducible Anti-Apoptotic Bcl Gene,
Inhibits Endothelial Cell Activation
By
Deborah M. Stroka,
Anne Z. Badrichani,
Fritz H. Bach, and
Christiane Ferran
From the Immunobiology Research Center, Beth Israel-Deaconess Medical
Center, Harvard Medical School, Boston, MA.
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ABSTRACT |
A1 is an anti-apoptotic bcl gene that is expressed in
endothelial cells (EC) in response to pro-inflammatory stimuli. We show that in addition to protecting EC from apoptosis, A1 inhibits EC
activation and its associated expression of pro-inflammatory proteins
by inhibiting the transcription factor nuclear factor (NF)- B. This
new anti-inflammatory function gives a broader dimension to the
protective role of A1 in EC. We also show that activation of NF-B is
essential for the expression of A1. Taken together, our data suggest
that A1 downregulates not only the pro-apoptotic and pro-inflammatory
response, but also its own expression, thus restoring a quiescent
phenotype to EC.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE VASCULAR endothelium in its quiescent
state promotes an anti-inflammatory and anticoagulant
environment.1 This phenotype changes significantly when
endothelial cells (EC) are subjected to pro-inflammatory stimuli such
as tumor necrosis factor (TNF) or lipopolysaccharide (LPS) in vitro or
in vivo, as can occur at sites of acute and chronic
inflammation.2-5 This phenotypic change, referred to as EC
activation, is characterized by transcriptional induction of a number
of pro-inflammatory and procoagulant factors such as adhesion
molecules, cytokines, and tissue factor.6 Even though this
pro-inflammatory environment promotes the generation of damaging levels
of reactive oxygen species and the activation of proteases, EC are
usually resistant to cell death.7-9
EC resistance is not likely related to the prototypic members of the
bcl family such as bcl-2 and
bcl-xL.10-12 Previous studies,
including our own, show that the constitutive expression of
bcl-2 (little to undetectable) and of
bcl-xL is not increased on stimulation of EC with
pro-inflammatory cytokines.13,14 The more recent discovery
of inducible anti-apoptotic genes in EC, such as the bcl gene
A1,13,15 the zinc finger protein A20,16,17 and
the newly described IAP genes,18 offers an explanation for the resistance of EC to inflammation-associated cell death.
A1 is a bcl family member that was originally identified as a
hematopoietic-specific early response gene induced upon stimulation with granulocyte-macrophage colony-stimulating factor.19
Further studies showed its expression in other cell types, ie,
EC.15 A1 expression was also found in tumors associated
with gastric cancers.20 In EC, A1 is induced through a
protein kinase C-dependent pathway in response to pro-inflammatory
stimuli and protects EC against TNF- and ceramide-mediated
apoptosis.13,15 We show here that the cytoprotective effect
of A1 is not limited to protection from apoptosis, but extends to
downregulation of EC activation. The inhibitory effect of A1 on EC
activation is due to inhibition of a key transcription factor required
for the up-regulation of most genes associated with EC activation,
namely nuclear factor (NF)-B.21,22
We further show that NF-B activation is required for A1 expression.
Inhibition of NF-B by overexpression of its specific inhibitor
IB ,23 abrogates TNF-mediated upregulation of A1. In
addition, A1 is induced in EC overexpressing p65. Thus, A1 is the first
bcl gene shown to depend on NF-B for its expression. This
observation suggests that NF-B is required not only for the
upregulation of the pro-inflammatory molecules associated with EC
activation, but also for the upregulation of "protective" molecules such as A1.
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MATERIALS AND METHODS |
Cell Culture
Human umbilical vein endothelial cells (HUVEC) were isolated and
cultured as described.24 Bovine aortic endothelial cells (BAEC) and the 293 human embryonic kidney cell line (ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) (GIBCO Life Technologies, Grand Island, NY) as described. All cells were cultured on tissue culture plates (Nunc; Marsh Products, Rochester, NY) precoated with 0.2% gelatin (Sigma Chemical Co, St Louis, MO) and
grown at 37°C in a humidified incubator with a 5% CO2 atmosphere.
Expression Plasmids and Reporter Constructs
A1 expression plasmid.
An N-terminally tagged A1 expression plasmid (HA-A1) was constructed by
reverse transcription-polymerase chain reaction (RT-PCR) from
TNF-induced HUVEC cDNA using the following primers: sense, 5'-TTGCTCGGATCCAGGCAGAAGATGACAG-3' and anti-sense,
5'-GTGTGAATTCTGGTCAACAGTATTGC-3'. The PCR product was cloned in the
pcDNA3 expression plasmid (Invitrogen, San Diego, CA)
tagged with the hemagglutinin (HA) nonapeptide sequence YPYDVPDYA (kind
gift of Dr J. Anrather, Immunobiology Research Center, Beth
Israel/Deaconess Medical Center, Boston, MA). Sequence
analysis of derived clones confirmed identity to the published human A1
sequence. The murine Bcl-2 cDNA is a kind gift of Dr T. Behrens
(University of Minnesota, Minneapolis). This cDNA was
subcloned in pAC expression vector. pAC is an 8.8-kb plasmid
vector containing a cytomegalovirus promoter,
a pUC19 polylinker site, and an SV40 splice/polyA site (a kind gift of Robert Gerard, University of Texas, Southwestern, Dallas, TX).
E-selectin, interleukin-8 (IL-8), ECI-6 (IB), pRc/RSV  eta
galactosidase (-gal), and NF-B reporters were previously
described.25 Human immunodeficiency virus (HIV)-long
terminal repeat (LTR) wild type (wt) and HIV-LTR  B
reporter (HIV)-chloramphenicol acetyltransferase (CAT) and
HIVB-CAT) are a kind gift of Ernst Böhnlein (Systemix, Inc,
Palo Alto, CA) and have been previously described; they represent
nucleotides  117 bp up to the TATA box of HIV-1 LTR for the
wt deleted from the NF-B binding sites (for the
B) cloned upstream of the CAT gene (CAT3N
polylinker).26
p65 Expression plasmid.
The p65 plasmid was a kind gift of Dr J. Anrather and represents human
RelA cDNA (amino acid 2-551) cloned in pcDNA3.
C-Tat is an expression vector encoding the HIV-1 Tat protein
(kind gift of Dr Ernst Böhnlein).26
Transient transfection assay.
2.105 BAEC were transfected with 1.5 µg/well of DNA
(expression plasmids and reporter constructs) using lipofectamine
(GIBCO) as previously described.25 In all experiments
(except HIV-CAT) 0.3 µg of the -gal reporter and 0.7 µg of the
E-selectin, IL-8, IB, or NF-B reporter was used with 0.001 to
0.5 µg of HA-A1 or pcDNA3 expression plasmids. In
experiments inducing the NF-B reporter by overexpression of p65
(RelA), 5 to 100 ng of p65 expression vector were added. For the
HIV-CAT and HIV B-CAT reporter
experiments, 0.2 µg of the -gal reporter was transfected with 0.5 µg of HA-A1 or pcDNA3, 0.2 µg of the c-Tat expression
plasmid, and 0.6 µg of the HIV-CAT or the
HIVB-CAT reporter. Cells were
stimulated 48 hours after transfection with either human recombinant
TNF (100 U/mL) (R&D, Minneapolis, MN) or LPS (100 ng/mL) (E
coli 0B55; Sigma), harvested after 7 hours and assayed for
-galactosidase, luciferase, or CAT activity, as previously
described.25 Luciferase and CAT activities were normalized
for -gal by using the formula: luciferase activity/-gal activity
×1,000. Normalized luciferase activity is given in relative light units.
Western blot analysis.
Cytoplasmic extracts were prepared from transfected BAEC and lysed in
RIPA buffer (10 mmol/L TRIS pH 7.5, 150 mmol/L NaCl, 1%
Triton X-100) containing 0.5 µg/mL of aprotinin, leupeptin, and
antipain, 0.5 mmol/L PMSF. Twenty µg of protein (determined by
Bradford assay; BioRad, Hercules, CA) were resolved on a reducing 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gel, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). Membranes were
blocked in BLOTTO (5% nonfat dry milk in 0.1% Tween 20 phosphate-buffered saline). HA-A1 protein expression was detected by
incubating 1/1,000 dilution of anti-HA antibody (Boehringer Mannheim,
Indianapolis, IN) followed by a 1/3,000 dilution of
peroxidase-conjugated goat anti-mouse secondary antibody (Pierce,
Rockford, IL) and revealed by enhanced chemiluminescence (ECL;
Amersham, Arlington Hights, IL).
Recombinant adenoviral-mediated gene transfer.
HUVEC were infected with a recombinant adenovirus (rAd) expressing
either A20 (gift of Dr V.M. Dixit, Genentech Inc, San Francisco, CA), porcine IB (gift of Dr C. Wrighton, Therexys
Ltd, Keele University, Keele, Staffordshire, UK),27 or, as
a control, -gal (gift of Dr R. Gerard), at a moiety of infection
(MOI) of 100 plaque-forming units/cell, previously shown to infect more
than 90% of the cells.27 HUVEC were assayed 48 hours after
the infection.
Northern blot analysis.
HUVEC, with or without rAd-mediated gene transfer, were stimulated for
3 hours with TNF (100 U/mL), LPS (100 ng/mL), or
phorbol-12-myristate-13-acetate (PMA) (5 × 10 8
mol/L) (Sigma). Total cellular RNA was extracted with Trizol (GIBCO)
and 10 µg of RNA were separated on a 1.3% agarose-formaldehyde gel
and transferred onto nylon membrane (Amersham). RNA was hybridized with
-[32P]-dATP (NEN) labeled probes as indicated
(Stratagene, La Jolla, CA). Membranes were stripped by boiling in 0.1%
SDS before reprobing with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) to correct for unequal sample loading.
RT-PCR.
BAEC were transfected with 1.5 µg of pcDNA3 or 0.9 µg
of pcDNA3 along with 0.6 µg of the p65 expression
plasmid. Eighteen hours after transfection, RNA was extracted with
Trizol. Five micrograms of total RNA were used for cDNA synthesis using
the superscript reverse transcriptase (GIBCO) and an oligodT primer. The cDNA were then amplified for A1 expression using primers derived from the human A1 sequence: sense primer 5'-AGATGACAGACTGTGAATTTGG-3' and anti-sense primer 5'-TGGTCAACAGTATTGCTTCAGG-3'. After an initial denaturation for 5 minutes at 94°C, the cDNA were amplified in a
Peltier thermocycler 200 (MJ Research Inc, Incline
Village, NV) for 35 cycles with each cycle programmed for a
denaturation at 94°C for 60 seconds, annealing at 50°C for 60 seconds, and elongation at 72°C for 60 seconds. The elongation time
of the last cycle was extended to 5 minutes. Amplified bovine A1 PCR product was sequenced using the DNA cycle sequencing kit from Perkin
Elmer (Norwalk, CT). To reliably compare the amount of amplified
product, cDNA samples were amplified with housekeeping GAPDH primers
which amplified products were recovered at 15, 20, 25, 30, and 35 cycles (Clontech, Palo Alto, CA).
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RESULTS |
A Novel Function for A1: Inhibition of EC Activation
To assess whether A1 could have an effect on EC activation, we used an
E-selectin reporter construct as a read-out for EC activation.
E-selectin is an EC-specific adhesion molecule that is upregulated in
response to pro-inflammatory stimuli.28 BAEC were
transfected with the E-selectin reporter together with either the HA-A1
expression plasmid or pcDNA3 in amounts ranging from 10 ng
to 0.5 µg. Transfection with increasing amounts of the HA-A1 plasmid
correlated with increased A1 protein expression as evaluated by Western
blot using an antibody against HA (Fig 1A).
Forty-eight hours after transfection, BAEC were stimulated for 7 hours
with TNF or LPS, then harvested and assayed for luciferase activity. In
BAEC transfected with pcDNA3, treatment with TNF and LPS
led, respectively, to 10- and 14.5-fold induction of E-selectin
reporter activity (Fig 1B, lanes 6 and 11 v 1). Coexpression of
A1 inhibited TNF- or LPS-induced luciferase activity in a
dose-dependent manner (Fig 1B, lanes 7 through 10 and lanes 12 through
15). Transfection of 0.5 µg of HA-A1 inhibited TNF-induced reporter
activity by 80% (Fig 1B, lane 10 v 6) and LPS-induced reporter
activity by 70% (Fig 1B, lane 15 v 11). Transfection with as
little as 1 ng of HA-A1 still inhibited reporter activity by 40% with
either TNF or LPS stimulation (Fig 1B, lane 7 v 6 and lane 12 v 11). In the next set of experiments, we chose reporters
constructed with promoters of genes that, like E-selectin, are
upregulated during EC activation: IL-8 and porcine IB (ECI-6). A1
expression inhibited the 3- and 4.5-fold induction of the IL-8 reporter
following stimulation with TNF or LPS (Fig 1C, lane 4 v 3 and
lane 6 v 5). Similar results were obtained with the IB
reporter. A1 expression inhibited the 2.5- and 3.5-fold induction of
the IB reporter following stimulation with TNF or LPS (Fig 1D,
lane 4 v 3 and lane 6 v 5).
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| Fig 1.
A1 expression inhibits E-selectin, IL-8, and IB
reporter activity. (A) Western blot analysis of A1 expressing BAEC. A1
protein was detected using anti-HA antibody at the expected molecular
weight of 20 kD, with as little as 0.001 µg (lane 2) of transfected
HA-A1 expression plasmid and expression was near optimal with 0.1 µg
(lane 4) (B) BAEC were transfected with the HA-A1 expression plasmid
titrated (0, 1 ng, 10 ng, 0.1 µg, or 0.5 µg) with
pcDNA3 to equal 0.5 µg of DNA together with the
E-selectin (0.7 µg) and -gal (0.3 µg) reporters. Forty-eight
hours after transfection, cells were stimulated with either TNF (100 U/mL) (lanes 6 through 10) or LPS (100 ng/mL) (lanes 11 through 15) for
7 hours. Results are given in relative light units. Data shown are
representative of six experiments. Error bars are ± SE (C and D) BAEC
were transfected with 0.5 µg of the HA-A1 expression plasmid together
with the IL-8 or IB (0.7 µg) and -gal (0.3 µg) reporters.
BAEC were then stimulated with TNF (100 U/mL) (lanes 3 through 4) or
LPS (100 ng/mL) (lanes 5 through 6). Results are given in relative
light units. Data shown are representative of three experiments.
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A1 Inhibits EC Activation Through Inhibition of NF-B:
Inhibition Occurs Upstream of p65-Mediated Transactivation
The transcription factor NF-B plays a key role in the up-regulation
of pro-inflammatory genes such as E-selectin, IL-8, and IB.21 Therefore, we questioned whether inhibition by
A1 of these reporters reflects an effect of A1 on NF-B activity.
BAEC were cotransfected with the HA-A1 expression plasmid in amounts ranging from 1 ng to 0.5 µg together with a reporter plasmid whose expression is strictly dependent on activation of NF-B. In BAEC transfected with pcDNA3, TNF and LPS, respectively, led to
a 30- and 60-fold induction of NF-B luciferase activity (Fig
2A, lanes 6 and 11 v 1). A1
expression inhibited TNF- or LPS-induced luciferase activity in a
dose-dependent manner (Fig 2A, lanes 7 through 10 and lanes 12 through
15). In the presence of 10 ng of the A1 expression plasmid, inhibition
of induction reached 60% with TNF or LPS stimulation (Fig 2A, lane 8 v 6 and lane 13 v 11). Transfection with 0.5 µg of A1
expression plasmid repressed TNF or LPS induction of NF-B reporter
activity by greater than 95% (Fig 2A, lane 10 v 6 and lane 15 v 11).
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| Fig 2.
A1 inhibits NF-B reporter activity. (A) Inhibition is
upstream of p65-mediated transactivation, (B) and is comparable to that
achieved with Bcl-2; (C) A1 expression does not inhibit the
NF-B-independent reporter, HIV- B CAT (D). (A) BAEC were
transfected with the HA-A1 expression plasmid titrated (0, 1 ng, 10 ng,
0.1 µg, or 0.5 µg) with pcDNA3 to equal 0.5 µg of DNA
together with the NF-B (0.7 µg) and -gal (0.3 µg) reporters.
Forty-eight hours after transfection, cells were stimulated with either
TNF (100 U/mL) (lanes 6 through 10) or LPS (100 ng/mL) (lanes 11 through 15) for 7 hours. Data shown are representative of six
experiments. (B) BAEC were cotransfected with 0.5 µg of
pcDNA3 or HA-A1 expression plasmid along with NF-B
reporter (0.7 µg), -gal reporter (0.3 µg) and in the presence of
increasing amounts of p65 expression plasmid ranging from 0 to 100 ng.
Data shown represent the fold induction of the NF-B reporter
luciferase activity by increasing amount of cotransfected p65. Data
shown are representative of three experiments performed. (C) BAEC were
cotransfected with 0.5 µg of pAC or murine Bcl-2 expression plasmid
along with NF-B reporter (0.7 µg), -gal reporter (0.3 µg),
and increasing amounts of p65 expression plasmid ranging from 0 to 100 ng. Data shown represent the fold induction of the NF-B reporter
luciferase activity by increasing amount of cotransfected p65. Data
shown are representative of two experiments performed. (D) BAEC were
cotransfected with 0.5 µg of pcDNA3 or HA-A1 together
with 0.6 µg of an HIV-B CAT reporter in the absence () or
presence of 0.2 µg (+) of the viral protein c-Tat. Results are
given in cpm ± SE. All error bars are ± SE. Data shown are
representative of three experiments performed.
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Having established that A1 interfered with NF-B activation, we
questioned whether this inhibition targets RelA/p65-mediated transactivation of NF-B. BAEC were cotransfected with 0.5 µg of
HA-A1 or pcDNA3 as well as the NF-B reporter construct.
Cotransfection of the p65 expression plasmid with amounts ranging from
5 to 100 ng induced a dose-dependent increase in the luciferase
activity of the NF-B reporter. A maximal induction of 40-fold was
achieved with the highest dose of p65 (100 ng). RelA/p65-mediated
induction of the NF-B reporter was not modified by expression of A1
(Fig 2B). This result indicates that A1 does not interfere with
p65-mediated transactivation in EC but rather exerts its effect further
upstream in the signaling cascade. Bcl-2 had a similar effect to A1 in EC; expression of Bcl-2 in BAEC did not affect the p65-mediated increase in the luciferase activity of the NF-B reporter (Fig 2).
Comparable induction of the luciferase activity was achieved in the
control pAC or the Bcl-2-transfected BAEC reaching again a 40-fold
increase with the highest p65 dose.
To determine whether the inhibitory effect of A1 on the activity of
NF-B-dependent reporters was selective, we evaluated whether A1
expression affects the induction of a c-Tat driven Sp1.26
Induction of the wt HIV-CAT reporter by coexpression of c-Tat was not
modified by A1 expression (data not shown). To further confirm that
NF-B binding sites were not involved in the induction of the HIV-CAT
reporter, a similar experiment was performed using the HIV
B-CAT reporter that has been
deleted from its two B binding sites but that still has all the Sp1
binding sites. Here again, A1 expression did not alter the 2.5- to
3-fold induction of the reporter observed on stimulation with c-Tat
(Fig 2D). Although much lower than the induction achieved in human cells, the 3-fold c-Tat-induced activation of the HIV-LTR reporter in
BAEC was consistently reproducible. Difference in induction might well
relate to interspecies variations. This result indicates that, in EC,
A1 does not have an overall inhibitory effect on transcription nor does
it inhibit the transactivation properties of Sp1.
NF-B Activation Is Required for A1 Expression in
HUVEC
To examine how the expression of A1 is regulated by pro-inflammatory
stimuli, RNA was extracted from HUVEC before and 3 hours after
treatment with 100 U/mL of TNF (lane 2), 100 ng/mL of LPS (lane 3) or 5 × 108 mmol/L PMA (lane 4). In consensus with recent
literature reports, Northern blot analysis of A1 mRNA showed that A1 is
not expressed constitutively, but is induced on stimulation with TNF or
PMA (Fig 3A).15 We show, in
addition, that A1 is induced following LPS stimulation (Fig 3A). In
contrast, constitutive mRNA levels of bcl-xL in EC,
were not modified by treatment with TNF, LPS, or PMA (Fig 3A).
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| Fig 3.
A1 expression is NF-B-dependent. (A) HUVEC were
stimulated with TNF (100 U/mL), LPS (100 ng/mL), or PMA
(5 × 108 mol/L) for 3 hours. A1 or
Bcl-XL mRNA was detected using
-[32P]-dATP-labeled specific cDNA probes. (B) HUVEC
were noninfected (NI) or infected with rAd.A20, rAd.IB or
rAd.-gal. Forty-eight hours after infection, HUVEC were either left
nontreated () or were stimulated with TNF (+) (100 U/mL). A1, A20,
and IB mRNA levels were detected by Northern blot analysis using
their specific -[32P]-dATP-labeled cDNA probes. GAPDH
was used to confirm equal quantity of RNA. Result shown is
representative of three independent experiments. (C) BAEC were either
nontransfected (NT) or transfected with a control plasmid
(pcDNA3) or the p65 expression plasmid (+p65). A1 mRNA
was only induced in p65 transfected BAEC (lane 5 v 3 and 4).
Lanes 1 and 2 correspond to negative (C) and positive controls (TNF
treated). GAPDH amplification (0.45 kb) was similar in all samples for
all cycles tested. Only the 20 cycles' amplification results are
shown.
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Most of the early phase response genes that are induced with EC
activation are dependent on activation of NF-B.21 To
assess whether NF-B is also involved in the induction of A1, HUVEC
cultures were infected with an rAd expressing IB, the specific
inhibitor of NF-B, at an MOI of 100. Forty-eight hours after
infection, HUVEC were stimulated with TNF for 3 hours, and total RNA
was extracted and assayed for A1 mRNA upregulation. A1 mRNA was not induced in response to TNF in HUVEC overexpressing IB (Fig 3B, lane 6 v 5), whereas high levels were detected in noninfected HUVEC (Fig 3B, lane 2 v 1), or in HUVEC infected with the
control rAd -gal (Fig 3B, lane 8 v 7). Comparably,
adenoviral-mediated gene transfer of A20, an anti-apoptotic gene that
inhibits activation of NF-B in porcine aortic EC, also resulted in
inhibition of A1 induction by TNF (Fig 3B, lane 4 v 3). To
confirm that in HUVEC A20 retains its inhibitory effect on NF-B
activation, electrophoretic mobility shift assay (EMSA)
analysis (data not shown) of nuclear extracts of HUVEC infected with
rAdA20 showed no NF-B binding activity following stimulation with
TNF.29
We then questioned whether p65 (Rel A) (the major transactivating
subunit within the Rel family members) was directly involved in A1 gene
induction. Total RNA was extracted from BAEC transfected with the p65
expression plasmid and the same amount (5 µg) from each sample was
then used for cDNA synthesis. Five microliters of the reverse
transcriptase reaction was then amplified by PCR for A1 or GAPDH
expression. Results showed that A1 mRNA expression is only induced when
p65 is overexpressed in BAEC but not in nontransfected or
pcDNA3-transfected cells (Fig 3C, lane 5 v 3 and
4). Positive and negative control were samples taken from quiescent and
TNF-stimulated BAEC (Fig 3C, lanes 1 and 2). The identity of the
amplified bovine A1 PCR product was confirmed by sequencing (data not shown).
| |
DISCUSSION |
EC are usually resistant to TNF-mediated death unless RNA or protein
synthesis is inhibited,9 which indicates that de novo protein synthesis is required to protect from apoptosis. A1, A20, and
the IAP proteins are TNF-inducible genes that belong to this category of newly synthesized anti-apoptotic proteins in
EC.13,15-18,30 We recently showed that A20 has a
broader cytoprotective role than merely inhibiting apoptosis;
overexpression of A20 also inhibits the up-regulation of genes
associated with EC activation by inhibiting the transcription factor
NF-B.25,29 A1 is a bcl family member that, unlike the
other anti-apoptotic bcl family members, ie, bcl-2 and
bcl-xL, is induced in response to pro-inflammatory stimuli in EC.15 The described function of A1 is to protect EC
against TNF- or ceramide-mediated apoptosis.13 More
recently, it has also been implicated as part of the cytoprotective
response of EC against LPS.31
In this paper we show that in EC, A1 has the same cytoprotective
properties as A20 ie protection from apoptosis and inhibition of
activation. As shown, overexpression of A1 inhibits the upregulation of
E-selectin, a specific marker for EC activation.32 In lieu of a protein measurement of E-selectin, this result was obtained using
a reporter containing the full porcine E-selectin promoter that has
been thoroughly characterized. This E-selectin reporter has been used
in several studies and shown to always correlate with the regulation of
the endogenous E-selectin.25,33-35 A1-mediated inhibition
of EC activation relates, at least in part and perhaps totally, to the
inhibition of the transcriptional factor NF-B. Like E-selectin, the
regulatory regions of most of the pro-inflammatory genes induced during
EC activation share a common feature; they contain at least one binding
site for NF-B.21 We also show that A1 equally suppresses
the expression of other NF-B-dependent pro-inflammatory genes ie,
IL-8 and IB. Taken together, these data suggest that A1
expression would markedly curb the inflammatory response associated
with EC activation.
The exact mechanism by which A1 inhibits activation of NF-B remains
to be determined. In EC, the major active form of NF-B is a
heterodimer of p50/NF-B1 and p65/RelA proteins. RelA/p65 contributes
to transcriptional activation whereas NF-B1/p50 is involved in DNA
binding.36 In quiescent EC, NF-B is held in the
cytoplasm by its inhibitory protein, IB.23
Stimulation of EC with agonists such as TNF, IL-1, PMA, or LPS initiate
a signaling cascade that results in the phosphorylation,37
ubiquitination,38 and degradation39,40 of
IB. NF-B is then able to translocate to the nucleus, bind to
its DNA sequence element, and initiate transcription of its target
genes. Our data shows that A1 does not interfere with p65-mediated
transactivation of NF-B; induction of NF-B reporter activity by
p65 expression is not altered by A1. This result contrasts with a
previous report studying Bcl-2. Bcl-2 was reported to inhibit NF-B
activation in 293 cells by downmodulating the transactivating potential
of nuclear p65.41 This difference does not relate to
variances in function between A1 and Bcl-2. We show here that, like A1,
expression of Bcl-2 in EC inhibited NF-B activation56
without altering p65-mediated transactivation. The difference in the
effect of the Bcl proteins upon p65-mediated transactivation is more
likely explained by cell-type specific function(s) of the bcl
genes as we were able to reproduce the inhibitory effect of Bcl-2 upon
p65-mediated transactivation in 293 cells (data not shown). Given that
the yield of transfected BAEC using lipofectamine does not usually exceed 10% to 15% of the cells, analysis of NF-B translocation to
the nucleus or of IB degradation following cytokine treatment could not be achieved. Generation of a replication defective adenovirus expressing A1 that would have the ability of transducing a high percentage of cultured EC is currently in progress and would help address these questions.
In summary, our data show that the inhibitory effect of A1 on NF-B
activation is at a level upstream of p65-mediated transactivation, however, the molecular basis of this novel function for A1 is still not
defined. One avenue of research is to evaluate whether the inhibitory
effect of A1 on NF-B activation requires its interaction with other
cellular proteins. Like Bcl-2 and Bcl-xL, A1 can
heterodimerize with pro-apoptotic bcl family members, namely
Bax.42 Whether interaction of A1 with Bax occurs in EC and
is equally as important for the inhibitory effect of A1 on NF-B
activation as for regulation of apoptosis needs to be tested.
Alternatively, A1 may interrupt the pathway leading to NF-B
activation by interacting with key signaling molecules. In favor of
this hypothesis are the data in the literature showing that Bcl-2
interacts with molecules such as p21Ras, p23Ras, Raf-1 kinase, and
calcineurin.43-47 For instance, the interaction between
Bcl-2 and calcineurin is critical for the inhibitory effect of Bcl-2 on
the transcription factor NF-AT (nuclear factor for activated T cells)
in T cells.47 One can speculate, giving the high homology
between Bcl-2 and A1, namely within the BH4 domain (necessary for all
interactions mentioned above), that A1 will also interact with similar
proteins to inhibit NF-B activation in EC. Further studies are
addressing this issue.
As previously stated, most of the early response genes associated with
EC activation are dependent on NF-B; this includes A20 whose
expression also requires NF-B.48,49 We questioned whether activation of NF-B is also a prerequisite for A1 expression. Using rAd, we overexpressed in HUVEC two inhibitors of NF-B: IB, the specific inhibitor of NF-B, or A20.25
HUVEC expressing either one of these inhibitors no longer upregulated
A1 mRNA after TNF stimulation. In lieu of a complete promoter analysis,
this result indicates that A1 expression, in EC, requires activation of
NF-B. The direct demonstration that A1 expression is induced in BAEC
by overexpression of RelA/p65 confirmed these data. A1 is, to the best
of our knowledge, the first bcl gene whose expression is dependent on
NF-B.
In conclusion, A1 appears to belong to the same category of
cytoprotective molecules in EC as the nonrelated anti-apoptotic protein, A20.50 When EC are confronted with
pro-inflammatory stimuli, cytoprotective genes are induced as part of
the activation process and help protect EC from apoptosis as well as
limiting EC activation through inhibition of the transcription factor
NF-B. These cytoprotective genes require NF-B for their
expression and therefore would downregulate not only the expression of
pro-inflammatory proteins, but also their own expression. This negative
feedback loop would then bring the cells back to their original
quiescent phenotype. That NF-B is necessary for inducing protective
proteins is supported by data from our own laboratory in EC, and others showing that inhibition of NF-B by overexpression of IB or by
knocking out p65/RelA sensitizes the cells to TNF-mediated apoptosis.51-54 It is clear that to achieve this
anti-inflammatory function, A1 and related proteins such as A20 need to
accumulate over time and probably attain a critical level to block the
activating pathway leading to NF-B activation. Further studies are
now performed to address this latter question, namely a tight study of
their kinetic of expression and their half life.
From a therapeutic point of view, blockade of NF-B has been
suggested as a possible approach to prevent pro-inflammatory consequences of EC activation, which have been implicated in several pathological conditions, including allografts and xenografts
rejection.2-5 To achieve this goal, a method to block
NF-B is needed that will not sensitize the cells to TNF-induced
apoptosis and even protect them against it. Indeed, EC loss will expose
the subendothelial matrix, which is equally as detrimental as the
consequences of EC activation itself. Expression of a gene such as A1
that inhibits inflammatory reactions and still protects the cells from
death may achieve this purpose. We propose that A1 will have the same potential as A20 of being a potent inhibitor of NF-B without sensitizing to TNF-mediated apoptosis,55 as occurs when
NF-B is inhibited with IB.51-54 Present studies
are addressing whether A1 and A20 have different intracellular targets
and thus would provide additive or synergistic protection in EC.
| |
ACKNOWLEDGMENT |
We thank Dr J. Anrather for his helpful discussions and resources,
namely the pcDNA3 HA-tagged expression plasmid and the p65
expression vector; Dr S.T. Grey for critical review of this manuscript;
Dr V. Dixit for providing the rAdA20, Dr Robert Gerard for the rAd
-gal control adenovirus; and E. Czismadia for her skilled technical
assistance in cell culture.
| |
FOOTNOTES |
Submitted October 5, 1998; accepted January 25, 1999.
D.M.S. and A.Z.B. contributed equally to this work.
This is manuscript 738 from our laboratories. F.H.B. is a paid
consultant of Novartis.
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.
Address reprint requests to Christiane Ferran MD, PhD, Immunobiology
Research Center, Beth Israel Deaconess Medical Center, 99 Brookline Ave, Boston, MA 02215; e-mail:
cferran{at}caregroup.harvard.edu.
| |
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ABSTRACT
INTRODUCTION
