Published online before print June 7, 2001, doi: 10.1161/hh1201.091796
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Overexpression of Ref-1 Inhibits Hypoxia and Tumor Necrosis Factor–Induced Endothelial Cell Apoptosis Through Nuclear Factor-
B–Independent and –Dependent Pathways
From the Division of Cardiovascular Medicine (J.L.H., G.H.G.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass, and Cardiovascular Research Institute (J.L.H., X.W., V.A., Y.Z., G.H.G.), Morehouse School of Medicine, Atlanta, Ga.
Correspondence to Jennifer L. Hall, PhD, Division of Molecular Cardiology, Department of Medicine, University of Minnesota, MMC, Mayo, 420 Delaware, Minneapolis, MN 55455. E-mail hallx{at}tc.umn.edu
Abstract
Abstract—
We hypothesized that a redox-sensitive transcription factor, redox
factor-1 (Ref-1) (HAP1, APE, and APEX), was critical in the regulation
of endothelial cell survival in response to
hypoxia and cytokines, including tumor necrosis factor
(TNF)-
. Hypoxia resulted in a significant decrease in Ref-1
protein expression in both human umbilical vein
endothelial cells and calf pulmonary artery
endothelial cells. The hypoxia-induced
decrease in Ref-1 expression was followed by a significant induction of
apoptosis as measured by caspase 3 activity and nuclear
morphology. Transient upregulation of Ref-1 significantly inhibited
hypoxia-induced apoptosis. However, deletion of the
redox-sensitive domain of Ref-1 abolished the antiapoptotic
effect. We postulated that the antiapoptotic effects of Ref-1
were mediated through nuclear factor-
B (NF-B). However, blockade
of NF-B with a dominant-negative IB (S32A/S36A) expression vector
had no effect on Ref-1–mediated survival under hypoxic conditions. The
second aim of this study was to test the cytoprotective ability of
Ref-1 upregulation in response to TNF-induced apoptosis. Ref-1
inhibition of TNF-induced death was associated with a significant
potentiation of NF-B activity. Deletion of the redox-sensitive
domain of Ref-1 significantly inhibited TNF-induced NF-B activation.
Moreover, loss of the redox-sensitive domain also abolished the
antiapoptotic effect of Ref-1 in response to TNF. To test
whether Ref-1 induced activation of NF-B was necessary to promote
survival, we blocked NF-B activity with a dominant-negative IB
(S32A/S36A). Indeed, blockade of NF-B activity abolished the ability
of Ref-1 to rescue TNF-induced apoptosis. In conclusion,
upregulation of Ref-1 promotes endothelial cell
survival in response to hypoxia and TNF through
NF-B–independent and NF-B–dependent signaling cascades,
respectively. Moreover, it seems that Ref-1 may act as a critical
cofactor, mediating the TNF-induced NF-B response in the vascular
endothelium.
Key Words: endothelium • apoptosis • hypoxia • redox • nuclear factor-B
Vascular endothelial cell apoptosis plays an important role in angiogenesis and vascular remodeling.1 2 3 4 5 6 7 8 Disruption of the endothelium in response to stroke, diabetes, ischemia, hypertension, and vascular injury is induced in part through activation of proapoptotic regulatory pathways.1 2 3 4 5 6 7 Moreover, recent evidence suggests that prevention of endothelial apoptosis may improve angiogenesis and endothelial function.6 8 Thus, defining novel genes expressed in the endothelium may lead to a better understanding of the signaling pathways mediating endothelial cell fate under conditions of angiogenesis and vascular remodeling.
Redox factor-1 (Ref-1) (also known as
APE,9
HAP1,10 and
APEX11 ) is a ubiquitous,
multifactorial protein that is a redox-sensitive regulator of multiple
transcription factors, including nuclear factor-B (NF-B), c-myc,
AP-1, and
HIF-19 10 11 12 13 14
as well as an apurinic/apyrimidinic endonuclease in the base excision
repair pathway. The potential importance of Ref-1 gene expression as a
critical determinant of cell fate is underscored by the fact that mice
lacking a functional Ref-1 gene die during embryonic development, with
embryos exhibiting a preponderance of pyknotic
cells.15 In addition, cell
lines expressing an antisense Ref-1 transcript exhibit a striking
increase in sensitivity to oxidative
stress.16 To our knowledge,
the role of Ref-1 as a determinant of endothelial cell
fate has not been described.
The endothelium plays a central role in
modulating the tissue response to ischemic injury. In the
context of ischemic injury, endothelial cell
viability may be compromised by hypoxia and the release of
cytokines by inflammatory cells. We hypothesized that Ref-1
plays a critical role in the regulation of endothelial
cell fate in response to pathophysiological
stimuli, such as hypoxia and tumor necrosis factor (TNF). The
present study demonstrates that the transient upregulation of Ref-1
significantly inhibits both hypoxia and TNF-mediated
endothelial cell apoptosis. Moreover, NF-B
activation seems to be a critical yet context-specific downstream
signal of Ref-1–mediated survival. Deletion of the redox-sensitive
domain of Ref-1 significantly inhibits NF-B activity and abolishes
this antiapoptotic effect. Taken together, our data suggest an
important role for Ref-1 in the vascular
endothelium.
Materials and Methods
Cells
Human umbilical vein endothelial
cells (HUVECs) (Clonetics) were grown in endothelial
growth media (Clonetics) that contains bovine brain extract (12
µg/mL), FBS (2%), hydrocortisone (1 µg/mL), human epidermal growth
factor (10 µg/mL), gentamicin (100 µg/mL), and amphotericin-B (0.05
µg/mL) and used between passages 2 and 5.
Calf pulmonary artery endothelial (CPAE) cells (ATCC) were grown in DMEM, 10% FBS, and 1% penicillin/streptomycin (Gibco, Life Technologies) and used between passages 18 and 26.
Materials
The following materials were used: human recombinant
TNF- (Genzyme), BCA protein assay (Biorad),
Hoechst 33342 (Molecular Probes) enhanced green
fluorescent protein expression vector (pEGFP-C1)
(Clontech), Mito tracker red expression vector
(pDsRed1-Mito) (Clontech), pCB6-Ref-1 (kind gift
from T. Curran, St. Jude Children’s Research Hospital, Memphis,
Tenn), IB dominant-negative expression vector (S32A/S36A)
(Upstate Biotechnology), pcDNA3.1 expression
vector (Invitrogen), a previously described
Ref-1 deletion mutant that abolishes all redox
activity,17
pNF-B–luciferase vector and thymidine-kinase-luciferase vector
(negative control) (Clontech), Effectene
(Qiagen), luciferase activity assay
(Promega), endothelial basal
medium, endothelial growth media, bovine brain extract
(Clonetics), protease inhibitor cocktail (Roche Molecular
Biochemicals), anti-Ref-1 (Santa Cruz Technologies), goat anti-rabbit
HRP-linked secondary antibodies (Transduction Labs), vimentin
monoclonal antibody (Sigma), chemiluminescent
detection reagent (Amersham Pharmacia Biotech)
caspase 3 cleavage activity assay (Biovision), and DEVD caspase 3
peptide blocker (DEVD-FMK) (Biovision).
Transfection
All transfections were carried out in CPAE
cells using a lipid-based transfection strategy (Effectene).
Transfection efficiency was 
25% to 30%. The previously described
Ref-1 deletion mutant lacking the N-terminal 116 amino acids that
abolishes all redox
activity17 was constructed
by amplifying 591 bp of Ref-1 with the following primers:
5'-GTAAAGCTTATGGATCAATACTGGTCAGCTCCTTCGG-3' and
5'-GTAGAATTCTTACAGTGCTAGGTATAGGGTGATAGG-3'. The product was
digested with HindIII and
EcoRI, sequence verified, and
cloned into pcDNA3.1.
CPAE cells were cotransfected with Effectene in the presence of growth media with one of the following expression vectors: pDsRed1-Mito or pEGFP-C1 (fluorescent markers) and pcDNA3.1 (control), pCB6-Ref-1, or a previously described Ref-1 deletion mutant.17
A subset of experiments involved cotransfection of
pCB6-Ref-1 or the control expression vector (pcDNA3.1) with an IB
(S32A/S36A) dominant-negative expression
vector.18 Twenty-four hours
after transfection, CPAE cells were exposed to hypoxic (2% oxygen) or
normoxic (21% oxygen) conditions while in the presence of growth media
(DMEM+10% FBS) for 24 hours. Alternatively, CPAE cells were treated
with human TNF in DMEM under normoxic conditions for 24 hours.
Apoptotic nuclei were assessed with Hoechst 33342 staining in
the transfected (pDsRed1-Mito or pEGFP-C1) population as previously
described.19
NF-B Activity
NF-B activity was determined by transiently
transfecting CPAE cells with the pNF-B–luciferase vector driven by
a herpes simplex virus thymidine kinase promoter or the thymidine
kinase–luciferase vector (negative control) along with pEGFP-C1 and
pCB6-Ref-1, the Ref-1 redox-sensitive deletion mutant, or a control
(pcDNA3.1) vector. The pNF-B–luciferase vector contains the
enhancer element within the promoter region. Once activated,
endogenous NF-B binds to the enhancer element and
activates luciferase expression. At 24 hours after
transfection, CPAE cells were exposed to hypoxia for 2, 4, 8,
and 24 hours under conditions of DMEM plus 10% FBS or treated with TNF
in DMEM (serum-free media) for 5 hours. Luciferase activity was
measured with a luminometer (Victor II, Wallac)
and normalized by assessing EGFP fluorescence (Victor II,
Wallac). All data are expressed as fold
activation of luciferase activity/EGFP fluorescence over
control-transfected cells under baseline conditions.
Initial experiments confirmed that transfection with a
control vector containing the herpes simplex virus thymidine kinase
promoter along with luciferase in the absence of the enhancer
elements had no significant effect on luciferase
activity.
Western Blotting
CPAE cells and HUVECs were lysed with RIPA buffer
(containing, in mmol/L, NaCl 150, Tris 10, EDTA 1, and PMSF 1 and
1% Triton X-100 and 1% deoxycholic acid)
containing a protease-inhibitor cocktail,
centrifuged, and assayed for total protein. Equal amounts of
cell lysates were loaded on 12% SDS gels, transferred to
nitrocellulose membranes, and probed as previously
described.19 As an
additional control, blots were reprobed with vimentin to confirm equal
loading as well as to verify that the decrease in Ref-1 protein
expression was not attributable to an overall decrease in protein
synthesis. Finally, all membranes were stained with Ponceau Red to
reverify equal protein loading and equal transfer of all protein to the
membrane.
Apoptosis
Both CPAE cells and HUVECs were grown on 6-well
plates to near confluence for all apoptosis experiments. For
the hypoxia experiments, CPAE cells were placed in DMEM plus
10% FBS and HUVECs were placed in endothelial basal
media supplemented with bovine brain extract. These media conditions
were chosen to minimize basal apoptosis. Both CPAE cells and
HUVECs were then placed in an incubator containing 2% oxygen
(hypoxia) or 21% oxygen (normoxia) for 24 hours.
It is noted that a separate series of experiments were completed in which HUVECs were also placed in DMEM plus 10% FBS (similar to CPAE cells) and subjected to hypoxia (data not shown). Results were similar under both media conditions.
For TNF-induced experiments, CPAE cells were placed in DMEM in the absence of FBS and HUVECs in endothelial basal media supplemented with bovine brain extract and treated with TNF or PBS for 24 hours. Apoptosis was assessed by staining with the nuclear chromatin dye H33342 and quantitating the percentage of apoptotic nuclei in each sample (400 cells counted per sample). As an additional assay of apoptotic cell death, caspase 3 activity was assessed using a fluorometric caspase 3 cleavage activity assay according to the manufacturer’s directions.19 Caspase 3 cleavage activity is expressed as a normalized ratio of arbitrary fluorophore units inhibitable by DEVD-FMK in treated cells divided by caspase activity in untreated cells. Our laboratory has performed extensive validations of these assays in vivo and in vitro with other techniques for assessing apoptosis.19 20 21
Statistics
Comparisons between two groups were analyzed
via Student’s t test
(P<0.05), and comparisons
between three groups were analyzed by ANOVA with a
Student-Newman-Keuls posthoc test
(P<0.05). n indicates the
total number of replicates in multiple experiments. Data are
presented as
mean±SE.
Results
Hypoxic Injury: Modulators of
Endothelial Cell Fate
Hypoxia induced a decrease in Ref-1 protein
expression in both CPAE cells and HUVECs
(Figure 1
). Ref-1 protein expression was significantly
decreased after 6 hours of hypoxia in CPAE cells
(Figure 1A) and at 18 hours in HUVECs
(Figure 1C). The differences in the time course of decay in
Ref-1 protein expression may likely be attributed to the different
origin of the cell types: pulmonary aortic (CPAE cells) or
umbilical vein (HUVECs). The loss of Ref-1 protein expression in
response to hypoxia was not attributable to a general decrease
in protein synthesis, because vimentin expression was unaffected
(Figure 1B).
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The hypoxia-induced decrease in Ref-1 protein expression was followed by a significant induction of apoptosis in CPAE cells and HUVECs at 24 hours as measured by assessing nuclear chromatin morphology and determining caspase 3 activity. In CPAE cells, hypoxia resulted in a significant increase in the percentage of apoptotic nuclei (normoxia, 5±1%; hypoxia, 15±1%; n=6, P<0.001). In accord with the significant increase in the condensed and coalesced nuclei, caspase 3 activity was significantly upregulated in CPAE cells after hypoxia (normoxia, 1.00±0.01 normalized ratio of arbitrary fluorophore units inhibitable by the DEVD caspase 3 blocker DEVD-FMK; hypoxia, 1.60±0.02; n=12, P<0.001). HUVECs also underwent apoptosis in response to hypoxia as measured by caspase 3 activity (normoxia 1.02±0.02; hypoxia, 1.13±0.01; n=12, P<0.001). We saw no significant change in the percentage of apoptotic nuclei at 8 or 16 hours in either the CPAE cells or HUVECs, suggesting that the decrease in Ref-1 protein expression precedes the induction of apoptosis.
We hypothesized that downregulation of Ref-1 may be
permissive in promoting apoptosis in response to
hypoxia. Accordingly, we examined whether upregulation of Ref-1
would modulate endothelial cell fate. The efficacy of
the transfection of Ref-1 is demonstrated by the significant increase
in Ref-1 protein expression
(Figure 2). In accord with our hypothesis, augmentation of
intracellular Ref-1 concentrations significantly inhibited
hypoxia-induced apoptosis in CPAE cells
(control-transfected, 26±2% versus Ref-1, 16±1%; n=8,
P<0.001)
(Figure 3).
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To begin to identify the region of Ref-1 responsible for this antiapoptotic effect, we used a previously described Ref-1 deletion mutant in which the well-defined redox-sensitive domain pertaining to the N-terminal 116 amino acids had been deleted.17 Interestingly, the redox deletion mutant abolished the antiapoptotic effect of Ref-1 under hypoxic conditions and, in fact, potentiated the death response (control-transfected, 26±2%; Ref-1, 16±1%; Ref-1 redox deletion mutant, 42±2%; n=8, P<0.001). Upregulation of the redox deletion mutant also resulted in significant potentiation of death under normoxic conditions (control-transfected, 17±1%; Ref-1, 12±2%; Ref-1 redox deletion mutant, 42±4%; n=8, P<0.001). These data suggest that endogenous Ref-1 likely plays a critical protective role in the endothelium. Moreover, they suggest that this Ref-1 redox deletion mutant may act as a dominant-negative construct, competing with and blocking endogenous Ref-1. Future studies will need to be completed to clarify this.
Ref-1 has been shown to activate
NF-B12 13 ;
thus, we hypothesized that the ability of Ref-1 to inhibit
hypoxia-mediated apoptosis was mediated through
potentiation of NF-B activity. This hypothesis was based in part on
previous studies demonstrating activation of NF-B in response to
hypoxia in endothelial
cells.22 23 24
In accord with our postulate, Ref-1 significantly potentiated NF-B
transactivation in CPAE cells with a luciferase-based reporter system
under hypoxic conditions (results expressed as fold activation over
control cells of luciferase activity normalized to EGFP
fluorescence) (control-transfected, 1.00±0.05;
Ref-1–transfected, 1.27±0.02; n=6,
P<0.001). However, under our
conditions of 2% hypoxia, NF-B was not significantly
activated above baseline in CPAE cells at multiple time points
(2, 6, 8 and 24 hours of hypoxia) (data not shown). In line
with this data, blockade of NF-B with a dominant-negative IB
construct did not have any effect on hypoxia-induced
apoptosis. Moreover, it had no significant effect on the
ability of Ref-1 to protect CPAE cells from apoptosis induced
by hypoxia; Ref-1 upregulation resulted in a 28±7% reduction
in apoptotic nuclei, whereas Ref-1 in the presence of the
dominant-negative IkB construct resulted in a 44±10% reduction. Taken
together, our data suggest that NF-B does not play a major role in
promoting endothelial cell survival in response to 2%
hypoxia and suggest that the survival-promoting effects of
Ref-1 in the setting of hypoxia are mediated through an
NF-B–independent pathway.
Cytokine Injury: Modulations of
Endothelial Cell Fate
To determine if Ref-1 was able to promote
endothelial cell survival in response to other
pathobiological stimuli present in the context of ischemic
injury, we exposed HUVECs and CPAE cells to human TNF for 24 hours. TNF
induced a dose-dependent apoptotic response in HUVECs as
measured by H33342
(Figure 4). TNF induced a similar dose-dependent effect in
CPAE cells (vehicle, 7±1%; 0.25 ng/mL TNF, 16±1%; 0.4 ng/mL TNF,
18±2%; 1 ng/mL TNF, 23±1%; 40 ng/mL TNF, 27±1%; n=10,
P<0.001). As another means of
confirming the apoptotic response, we measured caspase 3
activity. Indeed, caspase 3 activity was significantly enhanced in CPAE
cells treated with TNF (control, 1.00±0.02 versus 1.32±0.01; n=5,
P<0.001).
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Ref-1 gene expression was transiently upregulated in CPAE
cells to test our postulate that Ref-1 was an important mediator of
endothelial cell fate in response to TNF. Augmentation
of intracellular Ref-1 concentrations significantly inhibited
apoptosis induced by TNF (control-transfected, 43±2% versus
Ref-1–transfected, 22±2%; n=13,
P<0.001)
(Figure 5).
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In contrast to the response to hypoxia, it is
noteworthy that TNF did not alter Ref-1 protein expression in either
CPAE cells or HUVECs in a detailed time-course analysis (3 to
24 hours)
(Figure 6). This suggests that endogenous Ref-1
expression may be sufficient to maintain cell viability under normal
conditions but insufficient in response to the potent cytokine
TNF.
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On the basis of recent studies, we postulated that the
ability of Ref-1 to inhibit TNF-induced endothelial
apoptosis was attributable in part to activation of NF-B.
Treatment of CPAE cells with TNF induced a significant upregulation of
NF-B activity
(Figure 7). In accord with our hypothesis, augmentation of
Ref-1 expression resulted in a significant additive increase in NF-B
activity at baseline and in response to TNF
(Figure 7).
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To additionally delineate the role of NF-B as a distal
downstream signaling element critical to Ref-1–mediated survival in
response to TNF, we used a Ref-1 deletion mutant in which the
well-defined redox-sensitive domain pertaining to the N-terminal 116
amino acids had been
deleted.17 Loss of the
redox-sensitive domain abolished Ref-1–induced NF-B activation
(Figure 7
). These data suggest that Ref-1 may be a necessary
cofactor mediating TNF-induced NF-B activation. Moreover, the
cytoprotective effect of Ref-1 in response to TNF was abolished in
experiments involving the Ref-1 redox-sensitive deletion mutant
(control-transfected cells, 37±1% apoptotic nuclei;
Ref-1–transfected, 20±2%; Ref-1 deletion mutant, 51±4%; n=10,
P<0.001). It is noteworthy
that loss of the redox-sensitive domain potentiated the death response,
suggesting that the Ref-1 redox-sensitive domain plays an integral
protective role in the endothelium. These experiments
lend additional support to our hypothesis that Ref-1–mediated survival
in response to TNF was mediated in part through NF-B
activation.
Moreover, to directly test if potentiation of NF-B
activity was mediating the antiapoptotic signaling pathway
induced by Ref-1, CPAE cells were transiently cotransfected with the
dominant-negative IB (S32A/S36A) construct or an empty expression
vector along with Ref-1. Cells were treated with TNF, and
apoptosis was quantitated in the transfected subset. Initial
experiments confirmed blockade of TNF-induced NF-B activity
(control-transfected cells at baseline, 1.00±0.03; IB (S32A/S36A)
at baseline, 0.84±0.04; control-transfected+TNF, 2.69±0.29; IB
(S32A/S36A)+TNF, 1.3±0.07; n=3,
P<0.02). In accord with our
hypothesis, blockade of NF-B activation with IB (S32A/S36A)
abolished the ability of Ref-1 to inhibit TNF-induced apoptosis
(Figure 8). Thus, the data suggest that the ability of Ref-1
to inhibit apoptosis is attributable in part to its role in
potentiating the activation of NF-B.
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Discussion
Increasing evidence points to a role for
apoptotic regulatory pathways as critical determinants in the
progression of angiogenesis and vascular remodeling. Moreover,
prevention of endothelial apoptosis improves
angiogenesis after ischemia and preserves
endothelial dysfunction in disease states such as
diabetes.6 7 8
We have demonstrated a critical antiapoptotic role for the
multifactorial protein Ref-1 in endothelial cells in
response to hypoxia and TNF, two pathobiological stimuli
associated with ischemia-induced tissue injury. Transient
upregulation of Ref-1 was able to rescue endothelial
cells from both hypoxia and TNF-induced apoptosis.
However, deletion of the redox domain of Ref-1 abolished the
antiapoptotic effect, suggesting that this domain plays a
critical role in cytoprotection of the vascular
endothelium. Under hypoxic conditions, our data suggest
that Ref-1–mediated endothelial cell survival is
independent of NF-B activation. In contrast, Ref-1 potentiation of
an NF-B–signaling cascade seems to be critical in mediating the
antiapoptotic effect of Ref-1 in response to TNF. Thus, NF-B
activation seems to play an important yet context-specific role as a
downstream signaling target in the antiapoptotic function of
Ref-1 in the vascular endothelium.
Ref-1 is a multifactorial protein involved in both DNA
repair as well as redox-mediated transcriptional events, including the
activation of transcription factors NF-B, AP-1, c-myb, and members
of the ATF/CREB
family.12 13 17
The N-terminal 116 amino acid domain, partially absent in its bacterial
homolog exonuclease III, has been shown to be responsible for its redox
regulation, whereas the C-terminal domain is largely responsible for
its DNA repair activity.17
Several studies have documented that reducing agents, including
dithiothreitol, thioredoxin (upstream activator of Ref-1),
and Ref-1, stimulate DNA-binding activity of the p50 subunit of
NF-B.12 13 25 26
Moreover, Walker et al27
have identified a single cysteine residue (cysteine 65) as being the
redox-active site of Ref-1, thereby additionally substantiating that
the region of Ref-1 required to activate NF-B is located in
the N-terminal region. Our data suggest that loss of the redox domain
abolishes the antiapoptotic effects of Ref-1 in response to TNF
and hypoxia. Moreover, upregulation of the Ref-1 redox deletion
mutant actually potentiated death under these conditions as well as
under control conditions. Although speculative, our data would suggest
the likely possibility that this redox deletion mutant acts in a
dominant-negative manner. The increased susceptibility of
endothelial cells to both TNF- and
hypoxia-induced apoptosis after deletion of the redox
domain of Ref-1 suggests that endogenous Ref-1 plays a
critical protective role in the vasculature.
Hypoxia and TNF have both been shown to induce
NF-B activation in endothelial
cells.22 23 24 28 29 30
However, the role of NF-B under these conditions in promoting an
antiapoptotic signaling cascade is
controversial.24 28 29 30 31
We postulated that the antiapoptotic role of Ref-1 in response
to hypoxia and TNF was mediated through the potentiated
activation of NF-B. To test whether activation of NF-B was
necessary to promote Ref-1–mediated endothelial cell
survival, we used a genetic manipulation strategy of overexpressing the
dominant-negative IB (S32A/S36A) mutant that inactivates
NF-B.18 However, blockade
of NF-B had no significant effect on the ability of Ref-1 to prevent
hypoxia-induced apoptosis. These data suggest that
Ref-1 promotes endothelial cell survival under hypoxic
conditions via an NF-B–independent pathway. These data reconfirm
earlier work by Stempien-Otero et
al31 demonstrating that
hypoxia-induced endothelial apoptosis
is independent of NF-B.
In contrast, blockade of NF-B activation abolished the
ability of Ref-1 to protect endothelial cells from
TNF-induced apoptosis. As an additional critical test of our
hypothesis that Ref-1–mediated survival in the context of TNF was
mediated through NF-B, we used a Ref-1 mutant lacking the
redox-sensitive domain.17 In
accord with our earlier supporting evidence, the Ref-1 redox-sensitive
deletion mutant significantly attenuated NF-B activation in
endothelial cells. These data suggest that Ref-1 may
play a role as a critical cofactor mediating TNF-induced NF-B
activation. Taken together, our data suggest that Ref-1 mediates dual
antiapoptotic signaling cascades in endothelial
cells that are either independent or dependent on NF-B activation,
depending on the nature of the proapoptotic
stimuli.
Ref-1 is involved in the regulation of several transcription
factors in addition to NF-B that may be responsible for the
antiapoptotic effect of Ref-1 in response to hypoxia
and TNF-. Enhanced activation of AP-1 by Ref-1 has been described in
several cell
types.12 13 32
However, the role of AP-1 in regulating endothelial
cell fate is not
clear.33 34 Ref-1
has also been demonstrated to activate p53 and induce
apoptosis in human tumor cell
lines.35 Given that we
clearly document an antiapoptotic effect of Ref-1 on both
TNF- and hypoxia-induced apoptosis, it is unlikely
that a proapoptotic effect via p53 is a major component of its
effect in endothelial cells.
Inhibition of endothelial apoptosis
in response to hypoxia may have several therapeutic advantages.
Indeed, the regulation of a family of hypoxia-inducible factors
(HIFs) that bind specifically to promoters and enhancers in genes
important in adaptation to hypoxia, such as critical glycolytic
enzymes, glucose transporters, and vascular endothelial
growth factor, seems to play an important role in promoting
endothelial cell
survival.36 37
Interestingly, Ref-1 plays an important role in the regulation of
HIF-1
function.14 38 39
Upregulation of Ref-1 significantly potentiates hypoxia-induced
expression of a reporter construct containing the HIF-1–binding
site.14 Moreover, recent
work by Carrero et al38 and
Ema et al39 suggests that
Ref-1 is critical in the linking of two coactivator
proteins, CBP/p300 and SRC-1, to HIF-1. Complete loss of HIF-1
expression results in vascular malformations that correlate with cell
death and eventual embryonic
lethality.37 40
However, the influence of HIF-1 on cell fate is cell type–specific
and dependent on several factors, including growth factors, cell
density, pH, and glucose
concentrations.37 Thus, the
possibility that Ref-1 may inhibit hypoxia-induced
apoptosis through the regulation and stabilization of HIF-1
is intriguing. Our findings that deletion of the redox domain of Ref-1
abolished the antiapoptotic effect of Ref-1 additionally
suggest that this N-terminal domain is likely responsible for
biochemical interactions with several transcription factors mediating
survival, including HIF-1. Future studies will be needed to directly
address the role of HIF-1 and other transcription factors as
downstream mediators of Ref-1 signaling in hypoxic
conditions.
In conclusion, we have demonstrated for the first time that Ref-1 is a critical intrinsic factor promoting endothelial cell survival in response to pathophysiologically relevant stimuli. Indeed, the regulation of Ref-1 may play an important role in regulating vascular endothelial cell fate and function.
Acknowledgments
The authors gratefully acknowledge the grant support from the National Institutes of Health (J.H., G.G.), American Heart Association (J.H., G.G.), and the Pew Foundation (G.G.).
Footnotes
Original received October 26, 2000; resubmission received March 28, 2001; revised resubmission received April 20, 2001; accepted April 20, 2001.
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