(Received for publication, November 15, 1996)
From the Division of Cardiology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
The expression of the angiotensin II type 2 (AT2) receptor is developmentally and growth regulated. In cultured R3T3 cells, expression of this receptor is markedly induced at the confluent state and with serum deprivation. In this study we demonstrated that the removal of serum from culture media resulted in the induction of apoptosis in these cells and the addition of angiotensin II further enhanced apoptosis. We have previously identified an interferon regulatory factor (IRF) binding motif in the mouse AT2 receptor gene promoter region. In this report, we observed that serum removal increased IRF-1 expression, with a rapid and transient decrease of IRF-2. To prove that the changes in IRFs after serum removal mediated apoptosis and up-regulated AT2 receptor, we transfected antisense oligonucleotides for IRF-1 or IRF-2 into R3T3 cells and observed that IRF-1 antisense oligonucleotide attenuated apoptosis and abolished the up-regulation of AT2 receptor. IRF-2 antisense oligonucleotide pretreatment did not affect the onset of apoptosis after serum removal; instead, it increased AT2 receptor binding and enhanced angiotensin II-mediated apoptosis. Taken together, these results suggest that increased IRF-1 after serum starvation contributes to the induction of apoptosis and that increased IRF-1 up-regulates the AT2 receptor expression after serum starvation, resulting in enhanced angiotensin II-mediated apoptosis.
Angiotensin II (Ang II)1 exerts various actions in its diverse target tissues controlling vascular tone, hormone secretion, tissue growth, and neuronal activities. Most of the known effects of Ang II in adult tissues are mediated by the Ang II type 1 (AT1) receptor. Recently, a second receptor subtype known as AT2 receptor has been cloned by us and others (1, 2). The AT2 receptor is abundantly and widely expressed in fetal tissues, but present only in at limited levels in adult tissues such as adrenal medulla, specific brain regions, uterine myometrium, heart, and atretic ovarian follicles (3-8). The highly abundant expression of this receptor during embryonic and neonatal growth and quick disappearance after birth has led to the suggestion that this receptor is involved in growth, development, and/or differentiation. Our studies using in vivo gene transfer of the rat AT2 receptor into an injured rat carotid artery have demonstrated an attenuation of neointimal hyperplasia resulting from AT2 receptor transgene overexpression (9), and Stoll et al. (10) also demonstrated that the antiproliferative actions of the AT2 receptor offset the growth promoting effects mediated by the AT1 receptor.
Greater than 99.9% of the ovarian follicles present at birth undergo atresia, an event dependent on apoptosis or "programmed cell death." Interestingly, AT2 receptor expression is tightly associated with ovarian follicular atresia (3, 8). AT2 receptor is also abundantly expressed in immature brain and some specific regions of the brain (5, 6). Approximately half of the neurons produced during embryogenesis die by apoptosis before adulthood. These results have led us to examine the role of AT2 receptor in apoptosis. We recently demonstrated that the activation of the AT2 receptor induces apoptosis in a rat pheochromocytoma cell line (PC12W) and confluent mouse fibroblast cell line (R3T3 cell) (11). These cells have been shown to express abundant AT2 receptor (12-14). We further observed that higher AT2 receptor expression is especially associated with apoptosis (11).
In R3T3 cells, AT2 receptor expression is regulated by the growth state of the cells. AT2 receptor binding density is very low in actively growing cells, but increases markedly in the confluent, quiescent state (13, 14). We recently reported that the growth-regulated AT2 receptor expression in R3T3 cells is transcriptionally controlled by the competitive binding of interferon regulatory factor-1 (IRF-1) and IRF-2 for an IRF binding sequence in the AT2 receptor promoter region (15). IRF-1 and IRF-2 are structurally related, particularly in their amino-terminal regions that confer DNA binding specificity. They recognize the same DNA sequence elements (IRF-Es) (16, 17). IRF-1 functions as a transcriptional activator, whereas IRF-2 represses IRF-1 function by competing for the same cis-element (16-19). It has been shown that the growth state of certain cells depends on a balance between IRF-1 and IRF-2, i.e. a change in the IRF-1/IRF-2 ratio perturbs cell growth control (20-23).
In this study, we examined the role of AT2 receptor expression in IRF-regulated apoptosis. We hypothesized that changes in the ratio of IRFs under specific conditions regulate AT2 receptor expression which subsequently influences apoptosis. We studied the effect of serum starvation on R3T3 cells and observed that an increase in IRF-1 with transient decrease in IRF-2 stimulated AT2 receptor expression and enhanced AT2 receptor-mediated apoptosis.
R3T3 cells were cultured as described (13) using Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Cells were seeded at 7 × 104 cells/T-75 flask (Falcon) for transfection experiments for internucleosomal DNA fragmentation (DNA laddering), and cells were seeded on a 24-well plate (surface area, 2 cm2) (Falcon) at 2.5 × 104 cells/well for receptor binding studies. Two days after reaching confluent state, the cells were used for experiments. Cell number was counted by Coulter counter.
Oligonucleotides and Transfection to R3T3 CellsOligonucleotides used for treating cultured R3T3 cells as
"decoy" include the following: IRF binding,
5-GAAAAAGAGAAAGAGAAAATTCTGCTAAAAATGATA-3
mutant IRF binding,
5
-GAAAAAAGGAAAAGGAAAATTCTGCTAAAAAGGATA-3
. In mutant IRF-binding
oligonucleotide, three repeats of putative IRF binding hexamer motif as
previously reported (15) were replaced with C4 oligomer (AAAGGA), which
does not bind to IRFs (16). Oligonucleotides were annealed to
complementary sequences and used as double-stranded
oligonucleotides.
Phosphorothioate oligonucleotides for treating cells as antisense DNA
for IRF-1 and IRF-2 are as follows: IRF-1 antisense, 5-GAAAGATGCCCGAGATGC-3
(
111/
94) (24), IRF-2 antisense,
5
-GTGTGAGTGTTGTTAGGG-3
(
71/
54) (16).
On the day of transfection, the medium was changed to fresh medium and incubated for 1 h at 37 °C. The transfection was performed using LipofectAMINE Reagent (Life Technologies, Inc.), 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE), according to the manufacturer's instructions. Cells were incubated with the oligonucleotides (0.5 µM) and LipofectAMINE (1:3, w/w) for 3 h, and the transfection reagent was replaced with culture medium as described in each figure legend.
Receptor Binding AssayCells on the wells were washed twice
with 500 µl of phosphate-buffered saline containing 0.1% bovine
serum albumin. Reactions were performed with 0.2 nM
125I-CGP42112A (2,176 Ci mmol1, Peptide
Radioiodination Center, Washington State University) at 37 °C for
1 h. After incubation, cells were washed with ice-cold phosphate-buffered saline twice and solubilized with 200 µl of 0.5 N NaOH and solubilized material was counted for
radioactivity. Nonspecific binding was defined in the presence of 1 µM unlabeled CGP42112A.
Total RNA was prepared from cultured R3T3 cells
using RNAzol (Tel-Test), reverse transcribed using reverse
transcriptase and random hexamers (Perkin-Elmer), and applied to PCR.
PCR primers for IRF-1 used were as follows: 5-CCTGATGACCACAGCAGTTAC-3
and 5
-CTTCATCTCCGTGAAGACATG-3
. For IRF-2, PCR primers were
5
-GCGGTCCTGACTTCAGCTATA-3
and 5
-CTTCTTGATGACACTGGCCGG-3
. PCR
reaction was carried out with 30 cycles of 1 min f denaturation at
94 °C, 1 min of annealing at 55 °C, and 3 min of extension at
72 °C, followed by 15 min of final extension step. PCR-amplified
DNAs were subcloned into pCRTMII Vector (Invitrogen). cDNA probes
for Northern blotting were prepared from these plasmid vector by
restriction enzyme cut by EcoRI.
Total RNA was prepared from cultured R3T3 cells using RNAzol (Tel-Test) 24 h after oligonucleotide treatment. RNA (20 µg/lane) was separated by electrophoresis and transferred onto a nylon membrane (Amersham), and hybridization was carried out using a 32P-labeled IRF-1 and IRF-2 cDNA, a 32P-labeled HindIII-NsiI fragment of mouse AT2 receptor cDNA (25), or 0.78-kb PstI-XbaI fragment of a human glyceraldehyde-3-phosphate dehydrogenase clone. Densitometric analysis of autoradiograms was performed by scanning densitometer (GS300, Hoeffer) and NIH Image software.
Immunoblot AnalysisR3T3 cells were seeded onto 10-cm
dishes (Falcon) at 2 × 106 cells/dish for this
experiment. The cells were washed with HEPES-buffered saline and lysed
in 0.5 ml of phosphate-buffered saline containing 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate,
and 10 µg/ml aprotinin. Cell lysates were clarified by centrifugation
at 8,500 × g for 20 min, boiled in Laemmli loading
buffer for 3 min, resolved by 12% SDS-polyacrylamide gel
electrophoresis, electroblotted onto nitrocellulose membrane, and
immunoblotted with IRF-1 or IRF-2 antibody (Santa Cruz Biotechnology). Antibodies were detected by horseradish peroxidase-linked secondary antibody using the ECL (enhanced chemiluminescence) system
(Amersham).
DNA
extraction, subsequent 3 end-labeling of DNA, gel electrophoresis, and
quantitation of DNA fragmentation were performed as described
previously (26). Briefly, 500 ng of DNA prepared from each treated
cells was end-labeled with [
-32P]ddATP (Amersham) and
terminal transferase (Boehringer Mannheim) for 60 min at 37 °C.
Labeled DNA was loaded onto a 2% agarose gel, separated by
electrophoresis, and autoradiographed. The amount of radiolabeled ddATP
incorporated into low (<20 kb) molecular weight DNA fractions were
quantitated by cutting the respective fraction of DNA from the dried
gel and counting in a
-counter. The results were expressed as
percentage of the counts in control samples as described in each figure
legend.
R3T3 cells seeded onto six-well plates (Falcon) were used for this experiment. Chromatin binding dyes (Hoechst 33342 and propidium iodide (Molecular Probes)) were added to the serum-free medium at a concentration of 5 or 10 µM to examine the morphological changes of nuclei. After incubation at 37 °C for 1 h, cells were collected. After centrifugation, the pellet was resuspended in phosphate-buffered saline and cells were viewed under UV microscopy.
Statistical AnalysisAll values are expressed as mean ± S.D. Statistical significance was assessed by analysis of variance (ANOVA) followed by Scheffe's test. p < 0.01 was considered to be significant.
We first examined the changes
of IRF-1 and IRF-2 gene expressions after serum removal which induces
apoptosis in this cell line (11). As shown in Fig. 1,
expression of IRF-1 increased in a time-dependent manner
and showed a maximum at 24 h after serum removal, whereas IRF-2
mRNA transiently decreased 3 h after serum removal and
returned to basal level at 24 h. To document that these change in
IRF-1 and IRF-2 contributed to the increased AT2 receptor
expression, we treated confluent R3T3 cells with double-stranded
oligonucleotides spanning the IRF-Es in the mouse AT2
receptor promoter region (15) as transcriptional factor decoy, which
could bound with IRF-1 and IRF-2 in R3T3 cells (15). This strategy is
based on the competition for binding to a specific transcription factor
between a cis-element present in an endogenous target gene
and an exogenously added oligonucleotide corresponding to that
cis-sequence. One day after transfection of the
oligonucleotides, the cells were placed in serum-free medium, and
AT2 receptor binding was determined 3 days later by
radioligand receptor binding assay. As shown in Fig.
2A, serum depletion enhanced AT2
receptor binding. Pretreatment with IRF-binding decoy oligonucleotide
attenuated the increase of AT2 receptor in response to
serum removal. IRF-binding decoy oligonucleotide treatment did not
change the AT2 receptor density in serum-fed R3T3 cells.
Mutant IRF-binding decoy oligonucleotide treatment did not affect
AT2 receptor density. We also measured AT2
receptor mRNA levels at 24 h after serum removal and observed that pretreatment with IRF-binding decoy oligonucleotide inhibited the
AT2 receptor mRNA increase (Fig. 2B),
supporting the contention that the change in the ratio of IRF-1/IRF-2
after serum depletion is important for the transcriptional
up-regulation of AT2 receptor expression.
Effects of oligonucleotides spanning IRF binding sequence in mouse AT2 receptor promoter region and antisense oligonucleotides for IRF-1 and IRF-2 on AT2 receptor expression after serum starvation. Two double-stranded oligonucleotides (IRF and mutant IRF-binding decoy oligonucleotides) (see "Experimental Procedures") were transfected into confluent R3T3 cells, and 1 day after oligonucleotides transfection, serum was removed from the medium. The AT2 receptor binding was determined 3 days after serum removal by radioligand binding assay. A, the values were expressed as mean ± S.D. obtained from five different culture wells. Total RNA was prepared from R3T3 cells 24 h after serum removal or serum-fed cells. RNA (20 µg/lane) was separated by electrophoresis and hybridization was carried with a probe for the AT2 receptor or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Similar results were obtained in three different experiments (B). Antisense oligonucleotides for IRF-1 and IRF-2 were transfected into R3T3 cells. One day after oligonucleotide transfection, serum was removed from the medium. The AT2 receptor binding was determined 3 days after serum removal by radioligand binding assay (C). The values were expressed as mean ± S.D. obtained from five different cell culture wells. * shows p < 0.01 compared with LipofectAMINE transfection alone.
To prove that the changes of IRF-1 and IRF-2 expression after serum
depletion regulates the AT2 receptor expression, we
transfected antisense oligonucleotides for IRF-1 or IRF-2 into R3T3
cells 24 h before serum removal. To confirm whether these
antisense oligonucleotides inhibit IRFs expression, we examined the
IRFs protein expression 12 h after serum depletion. As shown in
Fig. 3, consistent with the mRNA expression of IRF-1
(Fig. 1A), we observed that IRF-1 protein was up-regulated
after serum depletion and IRF-1 antisense pretreatment inhibited
increased IRF-1 expression, whereas IRF-1 sense and IRF-2 sense and
antisense oligonucleotides did not influence the IRF-1 expression. On
the other hand, IRF-2 protein level was not changed 12 h after
serum depletion, although IRF-2 mRNA transiently decreased 3 h
after serum removal and returned to basal level rapidly. IRF-2
antisense oligonucleotide treatment decreased the expression of IRF-2.
Next we examined the effects of IRFs antisense oligonucleotides on
AT2 receptor expression and observed that IRF-2 antisense
enhanced AT2 receptor binding and IRF-1 antisense
oligonucleotide attenuated AT2 receptor binding in
confluent serum-fed R3T3 cells (Fig. 2C). We demonstrated
further that IRF-1 antisense oligonucleotide pretreatment abolished the up-regulation of AT2 receptor after serum removal (Fig.
2C), demonstrating that the increase in IRF-1 mediated the
up-regulation of AT2 receptor expression. In the
serum-depleted state, IRF-2 antisense treatment increased the
AT2 receptor density. These data suggest that increased IRF-1 and decreased IRF-2 after serum removal may exert synergistic effects on AT2 receptor up-regulation.
Induction of Apoptosis by IRF-1
To test the possibility that
the increased expression of IRF-1 in R3T3 cells after serum growth
factor removal contributes to the induction of apoptosis, we treated
the R3T3 cells with antisense oligonucleotides for IRF-1 or IRF-2 1 day
before serum starvation. As shown in Fig. 4, serum
depletion induced the internucleosomal cleavage of DNA, which resulted
in the generation of DNA fragments of multiples of ~180 base pairs
(the size of a nucleosome): a hallmark of apoptosis. IRF-1 antisense
oligonucleotide treatment inhibited DNA fragmentation in serum-starved
R3T3 cells, whereas IRF-1 antisense did not affect DNA fragmentation in
serum-fed R3T3 cells, suggesting that the increase of IRF-1 after serum depletion plays a critical role in the induction of apoptosis. On the
other hand, IRF-2 antisense oligonucleotide did not influence the
development of apoptosis. However, we cannot exclude the possibility that the transient decrease of IRF-2 mRNA after serum starvation as
shown in Fig. 1 enhanced the IRF-1 effect and consequently contributed
to the process of apoptosis as well. In addition to the DNA
fragmentation, we also examined the morphological changes of apoptosis.
As shown in Fig. 5, serum depletion caused apoptotic morphological changes in R3T3 cells. IRF-1 antisense oligonucleotide pretreatment attenuated the induction of apoptosis after serum growth
factor removal, consistent with the results of DNA laddering.
Possible Role of Up-regulated AT2 Receptor in the Induction of Apoptosis by IRFs
As shown in Figs. 6
and 7, Ang II (107 M)
treatment further enhanced DNA fragmentation after serum removal, and
this effect was blocked by the specific AT2 receptor
antagonist, PD123319 (Fig. 6, A and B). The
effect of Ang II on the induction of apoptosis was observed both in
serum-fed and serum-starved cells; however, the effect of Ang II was
significantly greater in serum-starved cells. Next we examined the
effects of IRFs on AT2 receptor-mediated DNA fragmentation
after serum removal (Fig. 7, A and B). Antisense oligonucleotides for IRF-1 and IRF-2 were transfected into R3T3 cells;
serum was removed from the culture medium 1 day after antisense transfection. The cells were then stimulated with Ang II
(10
7 M). Three days after Ang II stimulation,
DNA fragmentation was measured. IRF-1 antisense pretreatment inhibited
Ang II-mediated DNA fragmentation, whereas IRF-2 antisense pretreatment
further enhanced the effect of Ang II as a result of an increased
expression of AT2 receptor as shown in Fig. 2C.
Taken together these results suggest that the up-regulated
AT2 receptor density, as a result of the changes in IRF-1
and IRF-2, enhances the AT2 receptor-mediated apoptosis
after serum depletion.
The antigrowth effects of AT2 receptor on vascular smooth muscle cells (9) and endothelial cells (10) have been reported recently. The highly abundant expression of AT2 receptor during embryonic and neonatal growth, the rapid disappearance after birth (4, 6), and the up-regulation of AT2 receptor in myocardial infarction (27), cardiac hypertrophy (28), and skin wound (29) suggest that this receptor is closely involved with growth, development, and/or differentiation. Dudley et al. (14) reported that the addition of growth factors, such as basic fibroblast growth factor or serum, to quiescent R3T3 cells caused a rapid decrease in the number of surface AT2 receptor sites. Leung et al. (30) reported that nerve growth factor treatment decreased AT2 receptor expression in PC12W cells. Kambayashi et al. (31) also reported that the expression of AT2 receptor in cultured vascular smooth muscle cells was down-regulated by the addition of growth factors such as platelet-derived growth factor-BB, epidermal growth factor, and endothelin-1. Recently we have reported that this receptor induces apoptosis in PC12W cells by antagonizing the effect of nerve growth factor, and that apoptotic PC12W cells exhibited up-regulated AT2 receptor density (11). Moreover, we demonstrated that Ang II enhanced apoptosis in serum-starved R3T3 cells, along with the increased concentration of the AT2 receptor. These studies suggest that the expression of the AT2 receptor is inversely related to cell growth and is positively correlated with apoptosis. Consistent with our previous reports (11), we also demonstrated in this paper that AT2 receptor induces apoptosis in R3T3 cells; however, the "genetic evidence" that AT2 receptor induces apoptosis still remains to be proved.
To examine the molecular mechanism of AT2 receptor up-regulation in apoptosis, we studied R3T3 cells that express very low level of AT2 receptor in the growing state and abundant AT2 receptor after reaching the confluent state (14). To investigate the growth-regulated AT2receptor expression, we first cloned mouse AT2 receptor genomic DNA and studied its promoter function in R3T3 cells (15). We identified IRF binding motif (IRF-Es) in the promoter region of AT2 receptor gene and demonstrated that IRF-2 attenuated the AT2 receptor expression in both growing and confluent R3T3 cells, whereas IRF-1 enhanced AT2 receptor expression in the confluent cells only. Consistent with this result, gel mobility shift assay demonstrated that growing R3T3 cells exhibited only IRF-2 binding, whereas confluent cells exhibited both IRF-1 and IRF-2 bindings. In this paper, we demonstrated further that, in confluent R3T3 cells, the removal of serum increased IRF-1 expression but caused a rapid and transient decrease in IRF-2 mRNA expression. These changes of IRFs were associated with an up-regulation of AT2 receptor and the enhancement of AT2 receptor-mediated apoptosis. These results suggest that the IRFs play an important role in the up-regulation of this receptor associated with apoptosis.
IRF-1 and IRF-2 are structurally related, and they recognize the same DNA sequence elements AAGTGA motif or G(A)AAAG/CT/CGAAAG/CT/C (16, 17). Gene transfection studies have demonstrated that these two factors are mutually antagonistic; IRF-1 activates transcription, whereas IRF-2 inhibits it (16, 19). Harada et al. (23) have shown that the IRF-1/IRF-2 expression ratio oscillates throughout the cell cycle of NIH 3T3 cells, with IRF-1 expression at its highest when cells are growth-arrested owing to serum starvation and with IRF-2 being more abundant in growing cells. IRF-1 manifests antiproliferative properties (20, 22), whereas overexpression of the repressor IRF-2 leads to cell transformation and increased tumorigenicity, and this phenotype can be reverted by the concomitant overexpression of IRF-1 (23). Accordingly, we speculate further that the changes of IRF-1 and IRF-2 after serum removal are closely linked to the induction of apoptosis in R3T3 cells. Indeed our data demonstrated an increased IRF-1 expression and transiently decreased IRF-2 expression during apoptosis induced by serum removal.
Several lines of evidence has shown that the expression of oncogenes
can sensitize cells to undergo apoptosis, particularly under conditions
of low serum concentration or high cell density (32, 33). It has been
demonstrated that IRF-1 and IRF-2 have antioncogenic and oncogenic
activities, respectively. Deletion or inactivation of the IRF-1 gene at
one or both alleles have been detected in leukemia and myelodysplastic
syndromes (34). Tanaka et al. (35) reported that IRF-1 may
be a critical determinant of oncogene-induced cell transformation or
apoptosis in mouse embryonic fibroblasts (EFs). They demonstrated that
Ras signaling, under conditions of low serum or at high cell density or
following treatment by anticancer drugs or ionizing radiation, induces
the death of EFs derived from wild type mice and from mice with a null
mutation in the IRF-2 gene (IRF-2/
mice), but not of
EFs from IRF-1
/
and double knockout mice. These data
demonstrate that the absence of IRF-1 alone is sufficient to prevent
Ras-induced apoptosis. Tamura et al. (36) also showed that
the mitogen induction of the interleukin-1
-converting enzyme,
mammalian homologue of the Caenorhabditis elegance cell
death gene ced-3, is IRF-1-dependent. These
studies are consistent with our present results and support further
that IRF-1 is an unique transcription factor that functions as an
apoptotic inducer.
IRF-1 and IRF-2 are originally identified as regulators of type I interferon system. DNA sequences recognized by IRFs have been also found in the regulatory regions in a number of interferon-inducible genes (16-19, 37-39). IRF binding consensus element was also identified in inducible nitric oxide synthase (iNOS) gene promoter region, and IRF-1 is essential for iNOS activation in murine macrophages (40). Recently we reported that NO donor molecules, S-nitroso-N-acetylpenicillamine or sodium nitroprusside, induced apoptosis in cultured rabbit vascular smooth muscle cells (41). In this study, we observed that an increase in the ratio of IRF-1/IRF-2 after serum starvation mediated the up-regulation of AT2 receptor, resulting in the enhancement of Ang II-mediated apoptosis. Our data showed that the blockade of IRF-1 expression resulted in the simultaneous reduction in AT2 receptor expression and in apoptosis, whereas blockade of IRF-2 expression led to an increase in AT2 receptor expression and enhanced apoptosis. Taken together these data support the notion that AT2 receptor is one of the target genes of the IRFs system. We propose further that IRF-regulated gene products such AT2 receptor and iNOS may play important mediating roles in IRF-regulated apoptosis.
We gratefully acknowledge Dr. M. de Gasparo for CGP42112A, Dr. D. Dudley for R3T3 cells, and M. Hing for secretarial assistance.