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Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B), would prolong
survival of vascularized xenografts. Our previous studies have shown
that inhibition of NF-B by adenovirus-mediated overexpression of
IB
suppresses the induction of proinflammatory genes in EC.
However, IB sensitizes EC to TNF--mediated apoptosis,
presumably by suppressing the induction of the NF-B-dependent
anti-apoptotic genes A20, A1, manganese superoxide dismutase
(MnSOD), and cellular inhibitor of apoptosis 2. We report here that
adenovirus mediated expression of a dominant negative C-terminal
truncation mutant of p65/RelA (p65RHD) inhibits the induction of
proinflammatory genes, such as E-selectin, ICAM-1, VCAM-1, IL-8, and
inducible nitric oxide synthase, in EC as efficiently as does IB.
However, contrary to IB, p65RHD does not sensitize EC to
TNF--mediated apoptosis although both inhibitors suppressed the
induction of the anti-apoptotic genes A20, A1, and MnSOD equally
well. We present evidence that this difference in sensitization of EC
to apoptosis is due to the ability of p65RHD, but not IB, to
inhibit the constitutive expression of c-myc, a gene
involved in the regulation of TNF--mediated apoptosis. These data
demonstrate that it is possible to block the expression of
proinflammatory genes during EC activation by targeting NF-B,
without sensitizing EC to apoptosis and establishes the role of
c-myc in controlling induction of apoptosis during EC
activation. Finally, these data provide the basis for a potential
approach to suppress EC activation in vivo in transgenic pigs to be
used as donors for xenotransplantation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, bacterial LPS, IL-1-, IL-1-ß, or
IFN-
, activate EC. EC activation is characterized by induction of
proinflammatory genes, including those encoding adhesion molecules
(e.g., E-selectin, P-selectin, intercellular adhesion molecule-1
(ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)),
cytokines/chemokines (e.g., monocyte chemotactic protein-1, RANTES,
IL-1, IL-6, and IL-8), and procoagulant factors (e.g., tissue factor,
plasminogen activator inhibitor-1) (1, 2). Under
physiologic conditions the expression of these proinflammatory genes
plays a central role in triggering immune responses by selectively
recruiting circulating leukocytes to sites of inflammation. However,
uncontrolled expression of proinflammatory genes by EC can result in
vascular thrombosis and tissue necrosis such as observed during chronic
inflammation, ischemia-reperfusion injury, septic shock, and allograft
or xenograft rejection (2, 3, 4).
Induction of proinflammatory genes by EC is often triggered by
proinflammatory cytokines such as TNF- and is largely dependent on
the activation of members of the Rel nuclear factor B (NF-B)
family of transcription factors (5). In addition to the
signaling cascade leading to NF-B activation, TNF- activates
members of the Caspase family of proteases inducing apoptosis
(6). Most cells, however, do not undergo apoptosis when
stimulated by TNF-, likely based on the induction by TNF- of
anti-apoptotic genes such as A20 (7), the Bcl-2 family
member A1 (8), manganese superoxide dismutase (MnSOD)
(9) and cellular inhibitor of apoptosis-2 (c-IAP-2), a
member of the family of inhibitors of apoptosis (10). Most
of these anti-apoptotic genes, i.e., A20, MnSOD, and A1 also act as
inhibitors of NF-B and thus may control the expression of
proinflammatory genes during EC activation (3, 11, 12). We
refer to these anti-apoptotic genes as "protective genes" given
their dual role in protecting EC from apoptosis and blocking NF-B.
We have proposed a model for EC activation in which the expression of
protective genes down-modulates the proinflammatory response while
preventing EC apoptosis (3). We have shown that long term
survival of vascularized xenografts is associated with the expression
of protective genes by xenograft EC, whereas xenografts undergoing
rejection do not express these genes (3, 11).
Our hypothesis is that EC activation with expression of
NF-B-dependent proinflammatory genes underlies delayed xenograft
rejection by providing the proinflammatory environment necessary for
the activation and infiltration of recipient NK cells and monocytes,
and the development of thrombosis: hallmarks of this type of rejection
(4). Given the above, we have conceived strategies to
prevent xenograft rejection based on genetic engineering of EC and
aimed to inhibit EC activation through suppression of the transcription
factor NF-B (13). One such approach was to overexpress
IB, the natural inhibitor of NF-B (14). While
highly effective at blocking NF-B and suppressing the induction of
the proinflammatory genes, IB overexpression sensitized EC to
TNF--mediated apoptosis (14, 15), presumably by
inhibiting up-regulation of NF-B-dependent anti-apoptotic genes,
a finding that has been described in other cell types as well
(16, 17, 18). These findings, if also relevant in vivo, would
render this approach useless for xenotransplantation. An alternative
approach to block NF-B is based on the overexpression of a dominant
negative C-terminal truncation mutant of p65/RelA in EC
(13). This mutant, p65RHD, lacks the transactivation
domain of p65/RelA but retains the N-terminal Rel homology domain
(RHD), which is necessary and sufficient for DNA binding as well for
dimerization to other members of the Rel family (p50/NF-B1,
c-Rel/Rel) (13). We report here that recombinant
adenovirus-mediated overexpression of p65RHD in EC blocks NF-B
activity and inhibits proinflammatory gene induction equally as well as
does IB. However, contrary to IB, p65RHD does not sensitize
EC to TNF--mediated apoptosis, despite the fact that it blocks the
induction of the anti-apoptotic genes A20, A1, and MnSOD. We
explain this difference by the ability of p65RHD, but not IB, to
repress the expression of c-myc, a gene previously reported
to be involved in TNF--mediated apoptosis (19, 20, 21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Porcine aortic EC (PAEC) were isolated and grown on 0.2% bovine gelatin (Sigma, Saint Louis, MO)-coated cell culture flanks in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Life Technologies), penicillin G (50 U/ml), and streptomycin (50 µg/ml) (Life Technologies) as described before (22). Human umbilical vein EC (HUVEC) were isolated and grown in M199 medium (Life Technologies) supplemented with 15% FBS, NaH2CO3 (20 mM), HEPES (25 mM), glutamine (5 mM, Life Technologies), heparin (100 µg/ml), gentamicin (50 µg/ml), and endothelial growth factor (50 µg/ml) (Sigma) as described before (23). The murine 2F-2B cell line and the human 293 embryonic kidney cell line (American Type Culture Collection (ATCC), Rockville, MD) were grown in DMEM, 10% FBS, penicillin G (50 U/ml), and streptomycin (50 µg/ml).
Recombinant adenoviruses
The p65RHD-dominant-negative mutant has been described before
(13). Briefly, this construct encodes for amino acids (aa)
2–320 of the human p65/RelA preceded by a 13-amino acid sequence
containing 10 aa from the human c-myc gene used as a
recognition sequence by anti-C-terminal human
c-myc mAb 9E10 (hybridoma CRL-1729) (24). The
p65RHD-recombinant adenovirus was constructed as described before
for the IB-recombinant adenovirus (14). Briefly,
cDNA coding for p65RHD was cloned in to the pAC.CMV-pLpASR+
and the resulting p65RHD/pAC.CMV-pLpASR+ vector was
cotransfected into 293 cells with the pJM17 recombination plasmid
containing the full length adenoviral genome with a deletion in the E1
region. Adenovirus clones obtained by limiting dilution in 293 cells
were tested for p65RHD expression by Western blotting as described
below. The recombinant ß-galactosidase adenovirus was a kind gift of
Dr. Robert Gerard (University of Texas Southwest Medical Center,
Dallas, TX). The recombinant IB adenovirus expressing the porcine
IB gene (ECI-6) was generated in our laboratory by C. J.
Wrighton and has been described elsewhere (14). All
recombinant adenoviruses were produced in 293 cells, extracted,
purified through two cesium chloride gradient
ultracentrifugations, and their titer was determined by limiting
dilution in 293 cells as described before (14).
Adenovirus-mediated gene transfer to EC
Preconfluent PAEC were infected with a multiplicity of infection (MOI) of 500 plaque-forming units per cell (pfu/cell) and HUVEC were infected at 100 pfu/cell, respectively. Adenoviral infection was carried out in 1% FBS DMEM for 1.5 h at 37°C, 5% CO2, and 95% humidity under agitation. The FBS concentration was then adjusted to 10%, transduced EC were kept in presence of the recombinant adenovirus for 24 h, and the medium was replaced by fresh supplemented DMEM for an additional 24 h before being assessed for the expression and the function of the transferred gene. Unless otherwise indicated, all experiments were carried out with cells infected at the MOI giving maximal expression 2 days after infection.
Crystal violet vital staining
Cell viability was assessed by a colorimetric assay based on the
uptake of the vital dye, Crystal violet, as described elsewhere
(25). Briefly, cells were stained (2 min, room
temperature) with Crystal violet solution, washed under tap water, and
blue crystals were dissolved in 10% acetic acid (5 min, room
temperature). OD was measured at 
= 405 nm. For each recombinant
adenovirus infection, cells in culture medium were considered to
reflect 100% cell viability.
Transient transfection and plasmid constructs
The cDNAs encoding for human ß-galactosidase, p65, p65RHD and
a degradation-resistant mutant (26) of porcine IB
(ECI-6, a kind gift from Dr. R. de Martin, University of Vienna, Vienna
International Research Cooperation Center, Vienna, Austria) were cloned
into pcDNA3 vector (Invitrogen, Carlsbad, CA). The
DNA-binding-deficient mutants of p65 and p65RHD were derived through
PCR-based approach from p65 and p65RHD, respectively, as described
elsewhere (13, 27) and were expressed from the pCNDA3
vector. The full length human c-myc cDNA was cloned by PCR
from HeLa cell cDNA and expressed from the pcDNA3 vector. All
constructs were verified by dsDNA sequencing. All transient
transfection experiments were carried out using the murine EC line
2F-2B. All constructs were co transfected with ß-galactosidase and
the amount of DNA was maintained constant using the pcDNA3 vector.
Cells were seeded at 3 x 105 cells in 35-mm wells and
transfected 20 to 24 h later using Lipofectamine (Life
Technologies) according to the manufacturer’s suggestions (2–3 µg
DNA/6–9 µl Lipofectamine, 5–6 h). Twenty-four hours after
transfection, cells were stimulated with human rTNF- (100 U/ml,
16 h) (R&D Systems, Minneapolis, MN) and cell viability was
evaluated through detection of ß-galactosidase activity using 0.1%
X-Gal (Life Technologies) in 0.1 NaPi (pH 7.2), 1.3 mM
MgCl2, 3 mM potassium ferrocyanide, and 3 mM potassium
ferricyanide. Cell viability was assessed by counting the number of
"blue-stained" transfected cells that retained normal EC
morphology. The percentage of viable cells was normalized for each DNA
preparation to the number of transfected cells counted in the absence
of TNF- treatment. All experiments were performed three times in
duplicate.
Cell extracts and Western blot analysis
Cells were washed in PBS (pH 7.2), harvested by scraping,
centrifuged (300 x g, 5 min, 4°C) and total protein
was extracted (30 min, 4°C) in RIPA buffer (50 mM Tris, pH 8.0, 150
mM NaCl2, and 1% Nonidet P-40 (Sigma), 0.5% deoxycholate
(Sigma), 0.1% SDS supplemented with 0.1 mM TPCK (Sigma), 0.1 mM TLCK
(Sigma), 0.5 mM PMSF (Sigma), 1 µg/ml aprotinin (Boehringer Mannheim,
Indianapolis, IN), and 1 µg/ml Leupeptin (Boehringer Mannheim)).
Protein extracts were centrifuged (12,000 x g, 30 min,
4°C) and protein concentration was measured using the Bradford assay
(Bio-Rad, Hercules, CA). Equal amounts of protein were boiled in
Laemmli buffer, and electrophoresis was carried out under denaturing
conditions using 10% polyacrylamide gels. Proteins were transferred
onto a polyvinyldifluoridine membrane (Immobilon P, Millipore, Bedford,
MA) by electroblotting and detected using rabbit polyclonal Abs
directed against the N-terminal region of the human p65/RelA (Santa
Cruz Biotechnology, Santa Cruz, CA), the C-terminal region of human
c-myc (Upstate Biochemicals, Lake Placid, NY) or IB
(Santa Cruz Biotechnology). Proteins were visualized using horseradish
peroxidase-conjugated donkey anti-rabbit IgG (Pierce, Rockford, IL)
and the enhanced chemiluminescence assay (Amersham Life Science,
Arlington Heights, IL) according to the manufacturer’s
instructions.
Flow cytometry
EC were treated with human rTNF- (100 U/ml, 6 h) (R&D
Systems) and harvested by trypsin digestion (0.05% in PBS). Cells were
washed in PBS (pH 7.2), 5% FBS, 0.1% NaN3 and incubated
(1 x 102 cells in 100 µl, 30 min, 4°C) with a
mouse anti-human VCAM-1 mAb (Genzyme, Cambridge, MA), 1 µg/ml in
PBS, 5% FBS, 0.1% NaN3. After washing in PBS, 5% FBS,
0.1% NaN3 (300 x g, 5 min) cells were
stained (30 min, 4°C) with a FITC-labeled polyclonal goat
anti-mouse IgG Ab (Sigma). Fluorescent labeling was evaluated using
a FACsort equipped with Cell Quest Software (Becton
Dickinson, Palo Alto, CA). Specific labeling was compared with
nonspecific staining using a FITC-labeled isotype-matched control mAb.
Modulation of VCAM-1 expression was assessed by comparing the
expression on treated vs untreated cells. Detection of
phosphatidyl-serine in the outer leaflet of the plasma membrane of
apoptotic EC was carried out using FITC-labeled annexin V according to
the manufacturer’s suggestions (R&D Systems).
Cell cycle
PAEC were left untreated or infected with ß-galactosidase,
IB, or p65RHD-recombinant adenoviruses as described above and
analyzed 48 h after infection. Cell cycle progression was assayed
by propidium iodide DNA staining as described elsewhere
(28). Fluorescent labeling was evaluated using a FACsort
equipped with Cell Quest Software (Becton Dickinson). Experiments were
carried out in triplicate.
Immunocytochemistry
Confluent HUVEC or PAEC were trypsinized, centrifuged onto glass coverslips, fixed with 0.05% glutaraldehyde (15 min, room temperature), and permeabilized with Triton X-100 (Sigma). The presence of p65RHD was revealed by the anti-c-myc-specific mAb 9E10 using biotinylated goat anti-mouse IgG and horseradish peroxidase-coupled streptavidin (Pierce) as detection system for which AEC (3-amino-9-ethylcarbazole, Sigma) was used as substrate.
Northern blot analysis
PAEC and HUVEC were treated with TNF- (2 h, 500 U/ml) and RNA
was extracted using TRIzol, according to the manufacturer’s
suggestions (Life Technologies). Total RNA was separated on a 1.3%
agarose formaldehyde gel, transferred overnight to Hybond-N nylon
membranes (Amersham Life Science), and analyzed by specific
hybridization to radiolabeled cDNA probes for human A20 (kind gift from
Dr. V. Dixit, Genentech, South San Francisco, CA),
bcl-xL cloned in our laboratory by A. Badrichani, human
c-myc (as described above), MnSOD (ATCC), VCAM-1 (a kind
gift from T. Collins Brigham and Women’s Hospital, Boston, MA),
p65/RelA (a kind gift from W. C. Greene, University of California,
San Francisco, CA), rat inducible nitric oxide synthase (iNOS)
(29), porcine-E-selectin (30), intercellular
adhesion molecule-1 (ICAM-1), IL-8, and IB (ECI-6), as described
before (13, 31). All membranes were probed for
glyceraldehyde-3-phosphate dehydrogenase to control for equal RNA
loading.
Electrophoretic mobility shift assay (EMSA)
Nuclear proteins were extracted from HUVEC or 2F-2B EC by high
salt extraction as described before (13). All buffers were
supplemented with 0.1 mM TPCK, 0.1 mM TLCK, 0.5 mM, 1 µg/ml
aprotinin, 1 µg/ml leupeptin. Equal amounts of nuclear extracts (5
µg) were incubated (30 min at room temperature) with 100,000 cpm of
double-stranded, [-32P]dATP-radiolabeled
NF-B-specific oligonucleotide (5'-AATTTCAGAGGGGGATTTCCCAGAGG-3')
derived from the human light chain promoter or with
[-32P]ATP-radiolabeled NF-B oligonucleotide
(5'-AGTTGAGGGAATTTCCCAGGC-3') and the resulting DNA/protein
complexes were separated on a 5% polyacrylamide gel in
Tris/glycine/EDTA buffer at pH 8.5. Binding of AP-1 to the AP-1
DNA-binding consensus was carried out using 100,000 cpm of
double-stranded [-32P]ATP-radiolabeled AP-1
oligonucleotide (5'-TCTGACTCATCTCGA-3'). The mass of the probes was
approximately 20 fmol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and p65RHD-recombinant adenovirus
over expression
In order to achieve expression of p65RHD in vitro in essentially
all EC, we constructed a recombinant adenovirus for p65RHD, as
previously described for IB (Fig. 1
) (14). Transduction of
PAEC with increasing numbers of plaque-forming units per cell
(pfu/cell) of the p65RHD-recombinant adenovirus led to a dose-dependent
expression of p65RHD protein (Fig. 2A). Maximal expression was
obtained at a MOI of 500 pfu/cell (Fig. 2A) and of 100
pfu/cell in HUVEC (data not shown). p65RHD protein was detected 1 day
after infection and reached maximal levels at 2 days, declining
thereafter but persisting at significant levels at least until day 5,
the last time point analyzed (Fig. 2B). Adenovirus-mediated
transduction of p65RHD resulted in protein expression in the cytoplasm
and in the nucleus of virtually 100% of HUVEC, as analyzed by
immunocytochemistry (Fig. 2C). Similar results were obtained
in PAEC (data not shown).
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B
overexpression on IB degradation and endogenous NF-B DNA
binding following stimulation of HUVEC with TNF-. We have previously
suggested that p65RHD interferes with NF-B function by competitive
inhibition of DNA binding and have shown that expression of p65RHD does
not interfere with the degradation of IB (13). As
such, stimulation with TNF- resulted in rapid proteolysis of
IB (Fig. 3A) in
nontransduced ß-galactosidase or p65RHD-transduced HUVEC. This was
correlated with rapid nuclear translocation and DNA binding of
endogenous NF-B as tested by EMSA (Fig. 3B) and by
Western blot (data not shown). In HUVEC overexpressing IB, there
was no detectable IB degradation upon TNF- stimulation and,
consequently, no nuclear translocation of endogenous NF-B (Fig. 3A). Overexpression of p65RHD in HUVEC resulted in specific
DNA binding of p65RHD, which was not significantly altered following
TNF- stimulation (Fig. 3B). Presence of high levels of
p65RHD in these cells presumably competes with endogenous NF-B for
DNA binding since there was no detectable DNA binding of endogenous
nuclear NF-B in HUVEC transfected with p65RHD, while endogenous
NF-B was detected by Western blot (data not shown). Overexpression
of IB or p65RHD did not alter DNA binding of AP-1, another
transcription factor present in HUVEC (Fig. 3B).
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B at inhibiting the up-regulation of several NF-B-dependent
proinflammatory genes induced by TNF- in PAEC (Fig. 4). Induction of E-selectin, ICAM-1,
IL-8, and iNOS was inhibited by more than 90 to 95% upon
overexpression of either p65RHD or IB, as compared with
nontransduced or ß-galactosidase adenovirus-transduced PAEC.
Inhibition of endogenous IB induction, which is also NF-B
dependent, by p65RHD was also greater than 95% as compared with
nontransduced or ß-galactosidase adenovirus-transduced PAEC
(Fig. 4).
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B but not p65RHD sensitizes to TNF--induced apoptosis
Overexpression of either p65RHD or IB in HUVEC resulted in
total repression of proinflammatory gene induction by TNF- at both
the mRNA level (Fig. 4
) and at the protein level, as demonstrated for
VCAM-1 (Fig. 5A). Similar
results were obtained at the protein level for other proinflammatory
genes such as E-selectin (data not shown). Given the apparent equality
of p65RHD and IB in terms of inhibiting NF-B-dependent gene
expression, we tested the ability of these inhibitors to sensitize PAEC
to TNF--mediated apoptosis by analyzing translocation of
phosphatidyl-serine into the outer leaflet of the plasma membrane of
apoptotic EC, an early marker of EC apoptosis (32).
Transduction of PAEC with IB-recombinant adenovirus-sensitized
cells to TNF--mediated apoptosis, while nontransduced
ß-galactosidase or p65RHD-transduced cells did not undergo apoptosis
when stimulated with TNF- (Fig. 5A). Similar results were
obtained using a viability assay based on the staining of living EC
with Crystal Violet (Fig. 5B). PAEC transduced with p65RHD
did not undergo apoptosis when stimulated with TNF-, while PAEC
transduced with the IB-recombinant adenovirus did undergo
apoptosis (Fig. 5B). Similar results were obtained using a
transient transfection assay in 2F-2B EC line. Only about 30% of 2F-2B
EC transfected with IB survived TNF- challenge, whereas
essentially all cells transfected with the p65RHD or an empty control
plasmid survived the same treatment (Fig. 5C). Similar
results were obtained using HUVEC (Fig. 5B). However,
contrary to PAEC and 2F2B, HUVEC transduced with p65RHD showed some
level of cell death upon TNF- stimulation, which was, nevertheless,
threefold lower as compared with HUVEC transduced with the IB
adenovirus (Fig. 5B).
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B and p65RHD inhibit the induction of
anti-apoptotic genes
The presumed reason why expression of IB sensitizes EC to
TNF--mediated apoptosis is that NF-B-dependent anti-apoptotic
genes, such as A20, A1, MnSOD, and c-IAP-2, are repressed through
blockade of NF-B-mediated gene transcription. A differential effect
of IB and p65RHD on blocking the up-regulation of these
anti-apoptotic genes would explain the differential effects of
these two inhibitors on sensitizing EC to apoptosis. However,
overexpression of p65RHD was equally effective as compared with
IB at inhibiting the up-regulation of the NF-B-dependent
anti-apoptotic genes A20, A1, and MnSOD in HUVEC (Fig. 6). In addition, Stroka et al. in our
laboratories have recently shown that the inducible anti-apoptotic
gene, A1, is NF-B dependent and we have found that the induction of
A1 is equally well suppressed by p65RHD and IB (Fig. 6). The
expression of other anti-apoptotic genes such as
bcl-xL, which is constitutively expressed in HUVEC, was not
significantly affected by either p65RHD or IB overexpression
(Fig. 6). We have also tested the effect of p65RHD and IB
overexpression on the expression of the proapoptotic genes bax and bak,
which are constitutively expressed in HUVEC as tested by Western blot:
the expression of these genes was unaffected by either p65RHD or
IB overexpression (data not shown).
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-mediated apoptosis by
IB
To probe whether the lack of sensitization to TNF--mediated
apoptosis involved an active "protective mechanism" induced by
overexpression of p65RHD, we tested whether expression of p65RHD would
protect EC from sensitization to apoptosis by overexpressing IB.
Our hypothesis was that if overexpression of p65RHD was regulating an
anti-apoptotic mechanism (such as by altering the expression of a
given anti- or pro-apoptotic gene) then overexpression of p65RHD
should protect EC transfected with IB from undergoing apoptosis
when stimulated by TNF-. We first tested if cotransfection of full
length p65 with IB, at a ratio allowing nuclear translocation,
DNA binding, and transcriptional activity of p65, rescued EC from
TNF--mediated apoptosis. As illustrated in Figure 7A, p65
rescued EC-overexpressing IB from TNF--mediated apoptosis.
This result was consistent with both nuclear localization (Fig. 7B) and DNA binding (Fig. 7C) of p65, despite the presence of high levels of IB
in the cytoplasm (Fig. 7B). These data show that 2F-2B EC
behave in a manner similar to other cell types with respect to
induction of apoptosis by TNF- in the absence of NF-B
(16, 17, 18).
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B-dependent anti-apoptotic
genes and thus protects EC from TNF--mediated apoptosis. Transient
cotransfection of a DNA-binding-deficient mutant of p65 with IB
failed to protect EC from TNF--induced apoptosis (Fig. 7A). These data suggest that p65 DNA binding and/or
transcriptional activity are necessary to rescue EC from
TNF--induced apoptosis. We then tested the ability of p65RHD to
rescue 2F-2B cells overexpressing IB from undergoing
TNF--induced apoptosis. When overexpressed with IB, at a ratio
allowing nuclear localization and DNA binding, p65RHD rescued EC
overexpressing IB from TNF--induced apoptosis (Fig. 7). These
data show that the anti-apoptotic effect (protective activity to
apoptosis of p65RHD) is dominant over the IB-mediated
sensitization. The findings also suggest that the reason p65RHD does
not sensitize EC to apoptosis is not because it fails to block an
anti-apoptotic gene that is inhibited by IB. Rather, they
suggest that p65RHD has an additional active function as compared with
IB, and that it is this additional function that is responsible
for p65RHD-mediated protection from apoptosis. Transient cotransfection
of a DNA-binding-deficient derivative of p65RHD with IB failed to
protect EC from TNF--induced apoptosis (Fig. 7A). These
data suggest that the ability of p65RHD to protect EC from apoptosis
involves DNA binding but not transcriptional activation of
anti-apoptotic genes.
p65RHD, but not IB, represses basal c-myc
expression
In an effort to explain these findings, we studied the effects of
p65RHD and IB on expression of c-myc, which has been
implicated in the regulation of apoptosis in various cell types
(19, 20, 21). In our studies, overexpression of p65RHD
inhibited c-myc as studied at the mRNA level, whereas
IB did not (Fig. 8A).
This suppressive effect of p65RHD on c-myc expression was
dose dependent (Fig. 8B). It is possible that p65RHD
suppresses the expression of c-myc at the transcriptional
level through direct binding of p65RHD to NF-B consensus sites in
the c-myc promoter (33). Binding of p65RHD may
compete with other transcription factors involved in the basal
expression of this gene and thus would result in down-regulation of
c-myc mRNA expression. This hypothesis is currently being
tested.
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B is
inhibited sensitizes those EC to TNF--mediated apoptosis
Given that expression of c-myc in some cell types
correlates with the induction of apoptosis (19, 20, 21), we
questioned if the ability of p65RHD to suppress c-myc might
be responsible for protection from TNF--mediated apoptosis. The data
shown in Figure 9A support
this notion. Murine 2F-2B EC are resistant to TNF--induced apoptosis
when transiently transfected with empty plasmid (pCDNA3) or with
p65RHD. Expression of c-myc alone in 2F-2B cells failed to
sensitize to TNF--mediated apoptosis (Fig. 9A). However,
when both p65RHD, which inhibits up-regulation of the
anti-apoptotic genes (Fig. 6), and c-myc were
transiently overexpressed, 2F-2B EC were sensitized to TNF--mediated
apoptosis (Fig. 9A). This effect was dose dependent, i.e.,
higher amounts of c-myc induced higher levels of
sensitization to TNF--mediated apoptosis (Fig. 9B). We
conclude that c-myc expression may be necessary for the
induction of apoptosis by TNF- in EC under conditions in which
NF-B activity is repressed and anti-apoptotic genes are not
induced.
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B involved down-regulation of
c-myc. The data presented in Figure 9C
demonstrate that this is the case. Murine 2F-2B EC were transiently
transfected with IB and p65RHD, with or without ectopic
expression of c-myc. In the absence of c-myc
overexpression, p65RHD protected EC from sensitization to apoptosis by
IB (Fig. 9C). However, when c-myc was
overexpressed, EC became sensitive to apoptosis when challenged with
TNF- (Fig. 9C). This suggests that under conditions that
suppress NF-B, such as by overexpressing IB plus p65RHD,
expression of c-myc is necessary for the induction of
apoptosis by TNF- through a mechanism that we still do not
understand. This finding has potential general relevance to the use of
inhibitors of NF-B in situations in which apoptosis is
undesirable. Overexpression of p65RHD does not alter cell cycle progression in confluent EC
We tested whether suppression of expression of
c-myc protected EC from TNF--mediated apoptosis by
influencing a gene involved in cell-cycle regulation, which has been
demonstrated to be critical for induction of apoptosis in EC
(34); if p65RHD blocked progression through the cell
cycle, this would presumably protect EC from apoptosis. Given the above
we tested the effect of p65RHD overexpression on cell-cycle
progression. As expected, the majority of nontransduced confluent PAEC
was found to be in the G0/1 phase of the cell
cycle (>90%), as tested by nuclear propidium iodide staining (Fig. 10). Transduction of PAEC with
ß-galactosidase, IB, or p65RHD-recombinant adenoviruses did not
alter significantly the percentage of cells in each phase of the cell
cycle, i.e., Go/G1, S, G2/mitosis
(Fig. 10). These data indicate that differential sensitization of EC
transduced with IB or p65RHD to undergo apoptosis likely does not
involve differential cell-cycle progression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, and apoptosis has to date
been related primarily to the induction of anti-apoptotic genes.
This relationship is supported by findings of others that induction of
A20 (35), MnSOD (36) and, recently, c-IAP-2
are NF-B dependent (37), and results from our own
laboratories showing the same for A1 (Stroka et al., unpublished data).
To avoid the inflammatory response that is associated with organ
xenograft rejection, one would wish to inhibit NF-B, which we and
others have shown is key to the induction of the proinflammatory genes
in EC (14, 38). However, when IB is used to suppress
this aspect of EC activation, the EC are sensitized to TNF--mediated
apoptosis (Figs. 5 and 7) (15). Recent experiments
involving inhibition of NF-B by IB in other cell systems have
yielded similar results (17). Apoptosis of EC, which is
involved in the pathogenesis of septic shock (39) and is a
common feature of the pathology of xenograft rejection as well as
xenograft arteriosclerosis (15), is highly undesirable in
a clinical setting. Induction of EC apoptosis leads to cell retraction
and exposure of the subendothelium, which promotes platelet aggregation
and thrombosis. Furthermore, apoptotic EC have been shown to become
prothrombotic (32) and may activate complement through
C1q, further enhancing a prothrombotic environment
(40).
As an alternative to IB, we have developed a dominant negative
mutant of p65/RelA, one member of the Rel/NF-B family that is
intimately involved in proinflammatory gene induction during EC
activation. We refer to the mutant as p65RHD, since the C-terminal
(amino acid (aa) 321–551) transactivation domain was deleted, but the
RHD (aa 2–320) has been retained. The p65RHD mutant contains the
region from the wild-type p65/RelA that is responsible for binding to
IB. p65RHD retains the ability to bind to IB (data not
shown). Detection of high levels of nuclear p65RHD in nonstimulated
endothelial cells (Fig. 2) probably results from "saturation" of
the "available pool" of endogenous IB molecules present in the
cytoplasm of these cells (Fig. 2). Under these conditions, the
"excess" of p65RHD; which is not retained in the cytoplasm,
translocates to the nucleus (Fig. 2). In the presence of nuclear
p65RHD, DNA binding of endogenous NF-B is inhibited (Fig. 3). Given
that overexpression of p65RHD does not inhibit IB degradation
(Fig. 3) or nuclear NF-B translocation (data not shown), we conclude
that the presence of high levels of nuclear p65RHD competes with
endogenous NF-B for DNA binding, resulting in suppression of DNA
binding of endogenous NF-B.
Overexpression of p65RHD suppresses the transcriptional activity of
NF-B and inhibits the induction of proinflammatory genes equally as
well as does IB (13) (Figs. 4 and 5). However,
overexpression of p65RHD does not sensitize EC to TNF--mediated
apoptosis (Fig. 5) despite the finding that p65RHD, like IB,
suppresses the induction of the anti-apoptotic genes, A20, A1, and
MnSOD (Fig. 6) (Stroka et al., unpublished data).
There are at least two possible interpretations for the difference
between IB and p65RHD in terms of sensitizing EC to
TNF--mediated apoptosis. Expression of p65RHD may fail to suppress
one or more anti-apoptotic genes, known or unknown, which are
suppressed by IB, and thus EC would still remain resistant to
TNF--mediated apoptosis. Alternatively, it may be that p65RHD itself
exerts an additional function, as compared with IB, which results
in protection against apoptosis. To test these possibilities, we
coexpressed p65RHD and IB in EC and analyzed the ability of
TNF- to induce EC apoptosis. The fact that in this situation the
cells remained resistant to apoptosis (Figs. 5, 7, and 9) suggests that
the difference is not related to the failure by p65RHD to suppress
anti-apoptotic genes and supports the concept that p65RHD actively
provides protection. We have previously shown that overexpression of
p65RHD does not bind all of the available IB that is in the
cytoplasm, and allows IB to still prevent nuclear translocation
of the NF-B heterodimer, p50/p65 (13).
In an effort to understand possible anti-apoptotic mechanisms
that could be affected by p65RHD, we considered the relationship of
c-myc expression to apoptosis in EC. Clearly, p65RHD
overexpression in EC suppresses c-myc expression while
IB does not (Fig. 8A). To formally test the hypothesis
that c-myc must be present to allow TNF--mediated
apoptosis to occur in EC, we ectopically coexpressed p65RHD with
c-myc. The rationale for the experimental design was that
even though endogenous c-myc was suppressed by p65RHD, if
c-myc were present based on the transfection of the cells
with the c-myc vector, then the cells would become sensitive
to TNF--mediated apoptosis. Cells coexpressing c-myc and
p65RHD were sensitive to TNF-induced apoptosis, proving directly that
the effect of p65RHD on suppressing c-myc could by itself
account for the lack of sensitization by p65RHD to TNF--mediated
apoptosis (Fig. 9A). Furthermore, we tested whether the
mechanism by which p65RHD protected EC from sensitization to apoptosis
by IB also involved down-regulation of c-myc. In the
absence of c-myc expression, p65RHD protected EC from
sensitization to apoptosis by IB while ectopic expression of
c-myc under these conditions resensitized EC to apoptosis
(Fig. 9B). These data suggest that sensitization of EC to
apoptosis through blockage of NF-B requires the presence of
c-myc.
Our findings are consistent with the model that in the absence of
c-myc expression, the known NF-B-dependent inducible
anti-apoptotic genes (A20, A1, MnSOD) may not be required to
prevent apoptosis in EC. This hypothesis is consistent with the recent
observation that lymphocytes lacking the TNF receptor-associated
molecule TRAF-2 retain the ability to activate NF-B, and presumably
to induce the expression of NF-B-dependent anti-apoptotic genes,
but undergo apoptosis in the presence of TNF- (41, 42).
These authors thus suggest that NF-B-independent anti-apoptotic
genes may regulate induction of apoptosis by TNF-. Our data indicate
that, at least in EC, expression of c-myc regulates the
induction of apoptosis by TNF-, providing a "go-signal" that
allows the induction of apoptosis in the absence of NF-B-dependent
anti-apoptotic genes. The exact mechanism by which c-myc
promotes TNF--mediated apoptosis remains to be established. While
several reports have suggested that this mechanism may involve the
up-regulation of the proto-oncogene p53 (21, 43), it has
also been suggested that c-myc may be directly or indirectly
involved in the up-regulation and surface expression of the TNF
receptor family member, Fas ligand (FasL/CD95L) (44, 45).
In cells in which Fas (CD95) is constitutively expressed, up-regulation
of FasL would lead to apoptosis through Fas ligation (44, 45). However, ligation of Fas has been suggested to be
insufficient to induce apoptosis in EC (46) and thus
sensitization of EC to TNF--mediated apoptosis by c-myc
may not involve the Fas pathway. It has also been hypothesized that the
expression of c-myc in certain cell types may constitutively
down-regulate the expression of one or several anti-apoptotic genes
regulating the induction of apoptosis by members of the TNF receptor
family (44). Taking this into consideration,
down-regulation of c-myc would allow these
anti-apoptotic genes to be expressed and thus would be associated
with a protective phenotype. We favor this hypothesis as the mechanism
involved in protection of EC from TNF--mediated apoptosis by
c-myc down-regulation. Our preliminary data indicate that,
contrary to other cell types, c-myc expression in EC may not
be regulated by NF-B. This is supported by the observation that
c-myc is constitutively expressed in quiescent EC in the
absence of nuclear NF-B. Furthermore, TNF-, which mediates
nuclear NF-B translocation, does not up-regulate c-myc
expression in EC (data not shown). However, the c-myc
promoter contains two B-binding sites to which p65RHD may
potentially bind. It is therefore possible that binding of p65RHD to
these B sites may compete with other transcription factors involved
in the constitutive expression of c-myc in EC and thus would
result in c-myc down-regulation.
In conclusion, our data demonstrate that p65RHD is a dominant-negative
mutant that can be used to suppress NF-B specifically without the
undesirable effect of sensitizing EC to TNF--mediated apoptosis.
This makes p65RHD a candidate transgene that could be expressed in EC
of a porcine organ that is to be transplanted to a human. Expression of
p65RHD would prevent the response caused by induction of the
proinflammatory genes involved in the pathogenesis of delayed xenograft
rejection without the complications of rendering the EC susceptible to
apoptosis induced by TNF-, a cytokine present at essentially all
sites of inflammation.
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Acknowledgments |
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Footnotes |
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2 Current address: Zeneca Pharmaceuticals TDD/MCB 19F29
Mereside Macclesfield, Cheshire SK10 4TG, U.K.
3 Address correspondence and reprint requests to: Dr. J. Anrather, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02115. E-mail address
4 Abbreviations used in this paper: EC, endothelial cells; MnSOD, manganese superoxide dismutase; c-IAP-2, cellular inhibitor of apoptosis 2; NF-B, nuclear factor B; RHD, Rel homology domain; PAEC, porcine aortic endothelial cells; ICAM-1, intercellular adhesion molecule-1; MOI, multiplicity of infection; EMSA, electrophoretic mobility shift assay; pfu, plaque-forming units; FasL, Fas ligand; TPCK, L-p-tosylamino-2-phenylethyl chloromethyl ketone; TLCK, N--p-tosyl-L-lysine chloromethyl ketone.
Received for publication January 20, 1998. Accepted for publication June 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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