(Received for publication, May 11, 1995)
From the
Angiotensin II type 2 (AT) receptor is abundantly
and widely expressed in fetal tissues but present only in restricted
tissues in the adult such as brain and atretic ovary. This receptor is
speculated to be involved in tissue growth and/or differentiation. To
elucidate the molecular mechanism of growth-regulated AT
receptor expression, we cloned the mouse AT
receptor
genomic DNA and studied its promoter function in mouse
fibroblast-derived R3T3 cells, which express AT
receptor in
the confluent, quiescent state but very low levels of the receptor in
actively growing state. Promoter/luciferase reporter deletion analysis
of AT
receptor in R3T3 cells showed that the putative
negative regulatory region is located between the positions -453
and -225, which plays an important role in the transcriptional
control of AT
receptor gene expression along with the cell
growth. We identified the interferon regulatory factor (IRF) binding
motif in this region using DNase footprinting analysis and demonstrated
that IRF binding oligonucleotide treatment increased the AT
receptor expression in growing R3T3 cells but not in confluent
cells. Furthermore, by antisense treatment, we demonstrated that IRF-2
attenuated the AT
receptor expression in both growing and
confluent R3T3 cells, whereas IRF-1 enhanced AT
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 binding. Furthermore, we observed using reverse
transcription-polymerase chain reaction that the IRF-1 mRNA expression
was more abundant in confluent cells than growing cells, whereas IRF-2
expression did not change with R3T3 cell growth. We conclude that, in
confluent cells, the enhanced expression of IRF-1 antagonizes the IRF-2
effect and increases the AT
receptor expression. We
speculate that these transcriptional factors influence cell growth in
part by regulating AT
receptor expression.
Angiotensin II (Ang II), ()a key regulator of
cardiovascular homeostasis, exerts various actions in its diverse
target tissues controlling vascular tone, hormone secretion, tissue
growth, and neuronal activities. Multiple lines of evidence have
suggested the existence of Ang II receptor subtypes, but it was only
recently that at least two distinct receptor subtypes were defined.
Based on their differential pharmacological and biochemical properties,
the subtypes have been designated as type 1 (AT
) and type 2
(AT
) receptors(1, 2) . To date, extensive
pharmacological evidence indicates that most of the known effects of
Ang II in adult tissues are attributable to the AT
receptor. In contrast, much less is known about the regulation
and function of the AT
receptor. It is abundantly and
widely expressed in fetal tissues and immature brain (3, 4, 5) but present only in scant levels in
tissues in the
adult(1, 2, 3, 5, 6, 7) ,
suggesting that this receptor may be involved in tissue growth and/or
differentiation. Using the expression cloning strategy in COS-7 cells
from a rat fetus expression library, we have reported the cloning of
the rat AT
receptor(8) ; Kambayashi et al.(9) also cloned rat AT
receptor from rat
pheochromocytoma cell line PC12W cells. Hydropathy analysis indicates
that it belongs to a seven-transmembrane receptor family. It is 34%
identical in sequence to AT
receptor. The signal mechanism
of the AT
receptor is still poorly defined.
To
understand the molecular mechanism of the developmental and growth
regulation of AT receptor expression, we cloned, in this
study, mouse AT
receptor gene, analyzed its structure, and
examined the promoter activity. We then employed R3T3 cells, a mouse
fibroblast cell line, as model to study the AT
receptor
promoter activity since these cells express the only AT
subtype binding sites, and the expression of AT
receptor sites in these cells are modulated by the growth state
of the cells (10, 11) . This characteristic of this
cell line provides us an excellent model for studying the growth
regulation of the AT
receptor.
Interferon regulatory
factor (IRF)-1 and related IRF-2 protein have been shown to recognize
same DNA sequence elements(12) . IRF-1 activates transcription
of interferon (IFN)-inducible genes, whereas IRF-2 inhibits
it(12, 13, 14, 15, 16, 17) .
Recently, competitive binding of IRF-1 and IRF-2 was reported to play
critical roles in determining cell growth, transformation, and
apoptosis. Furthermore, IRF-1 has been implicated to be a tumor
suppresser gene and IRF-2 as an
oncogene(18, 19, 20) . Our results show that
the expression of AT receptor in R3T3 cells is
transcriptionally regulated by the competitive binding of IRF-1 and
IRF-2, suggesting that AT
receptor is one of the target
genes of IRF system and that IRF system may regulate the cell growth
via the AT
receptor.
Figure 1:
Sequence analysis of promoter region of
mouse AT receptor (A) and putative transcription
factor binding sites (B). Restriction enzyme sites for making
AT
receptor promoter-luciferase expression vectors (XhoI, HaeIII, PvuII, DraI, and HindIII) and transcription initiation sites (P1, P1`, and P2)
are shown. The transcription initiation site P1 is presented as
position 0. Bold and italic nucleotides indicate the
sequence of primer for primer extension
analysis.
IRF binding oligonucleotide was also used in gel mobility shift assay. IRF and octamer binding sequences were designed according to the footprinted sequences (Fig. 3, A and B). Three repeats of putative IRF binding hexamer motif, as shown in Fig. 1B, were replaced with C4 oligomer (AAAGGA), which does not bind to IRFs(14) , and this oligonucleotide was used as mutant IRF binding oligonucleotide. Oligonucleotides were annealed to complementary sequences and used as double-stranded oligonucleotides. Oligonucleotides for treating cells as antisense DNA for IRF-1 and IRF-2 were as follows: IRF-1 antisense, 5`-GAAAGATGCCCGAGATGC-3` (-111/-94)(25) ; IRF-2 antisense, 5`-GTGTGAGTGTTGTTAGGG-3` (-71/-54)(14) .
Figure 3:
DNA binding properties of negative
regulatory regions by DNase I footprinting (A and B),
the effect of oligonucleotides spanning two distinct protected regions
observed by DNase I footprinting on AT receptor binding (C and D), and expression in R3T3 cells (E). A and B, probe DNAs AccI-DraI
(-443/-224) (A) and HinfI-DraI
(-340/-224) (B) fragments were subjected to DNase
I footprinting analysis. Lanes1 and 2,
probe DNAs were reacted with nuclear extracts prepared from growing
cells (15 and 30 µg, respectively); lanes3 and 4, probe DNAs were reacted with nuclear extracts prepared from
confluent cells (15 and 30 µg, respectively) and digested by DNase
I. LanesG and G+A, probe DNA cleaved
by G and G + A reactions(41) . The protected regions are
depicted on the leftsides of the panel, and
the non-protected region is underlined. C and D, three double-stranded oligonucleotides (octamer binding,
IRF binding, and mutant IRF binding oligonucleotides) (see
``Experimental Procedures'') were transfected into growing (C) and confluent (D) R3T3 cells. The AT
receptor binding in untreated cells is taken as 100%. The values
were expressed as mean ± S.D. obtained from eight different cell
culture wells. E, total RNA was prepared from cultured R3T3
cells 24 h after oligonucleotide treatment. RNA (20 µg/lane) was
separated by electrophoresis, and hybridization was carried with a
probe for the AT
receptor or for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as a control. The result is a
representative of data obtained in three different
experiments.
We screened mouse liver genomic libraries by PstI-NsiI fragment of mouse AT receptor
cDNA (21) and obtained three independent positive clones.
Restriction enzyme mapping revealed that these three clones overlapped
each other, and each clone contained full-length mouse cDNA. One of
these (MG1, 14.3 kilobases) was further analyzed. Sequence comparison
between genomic DNA and cDNA reveals that there are two introns in the
5`-untranslated region. Coding region in the third exon is not
interrupted by any intron, and there is no intron in the
3`-untranslated region. Similar observation was reported by Ichiki et al.(29) using 129SV mouse strain. To investigate
further the role of the promoter region in controlling AT
receptor gene expression, we sequenced the upstream promoter
region (Fig. 1) and performed primer extension analysis using
mouse fetus RNA (data not shown). Primer was designed in the upstream
of the first intron as shown in Fig. 1. Primer extension
products were observed at two distinct sites (P2 and P1, P1`) (Fig. 1A). P1 and P1` corresponds to T and G, and there
are 3 bp between these initiation sites. Adjacent to these initiation
sites, a TATA motif is observed in the upstream sequence. Another
primer extension product (P2) is observed at position -67. In the
following experiments, we assigned P1 site as the initiation site to
the sequence.
To investigate the growth-dependent AT receptor expression, we used R3T3 cells. Specifically, the
expression of AT
binding sites is very low in actively
growing cells but markedly increased in confluent, quiescent
cells(11) . We subcloned HindIII-XhoI
fragment (-1081/+52) of MG1 into the enhancerless luciferase
expression plasmid (p0Luc) and transfected this construct into the R3T3
cells. Luciferase activity obtained in experiment with
p(-1081/+52)Luc using confluent R3T3 cells was taken as 100%
in each experiment. Transfection with p0Luc gave a background
luciferase activity. As shown in Fig. 2, A and B, luciferase expression by this AT
receptor-luciferase expression vector construct was 5-fold higher
in the confluent R3T3 cells than that in the rapidly growing cells.
Transfection with pSVLuc, which contains p0Luc plus the heterologous
SV40 virus early promoter/enhancer region driving luciferase
expression, did not show the difference in luciferase activity between
growing and quiescent R3T3 cells. These results suggest that the
AT
receptor promoter region (-1081/+52) contains
specific cis DNA elements that determine cell growth-regulated
AT
receptor gene expression.
Figure 2:
Transcriptional activity in R3T3 from
deletions of the mouse AT receptor promoter region. The
chimeric mouse AT
receptor promoter-luciferase constructs,
p(-1081/+52)Luc, p(-892/+52)Luc,
p(-802/+52)Luc, p(-453/+52)Luc,
p(-224/+52)Luc, p0Luc, and pSVLuc were transfected in
growing R3T3 cells (A) and confluent, quiescent R3T3 cells (B). Luciferase activity obtained in experiment with
p(-1081/+52)Luc using confluent R3T3 cells was taken as
100%. pSV-
-galactosidase control vector was cotransfected to
normalize variations in transfection efficiency. The values (mean
± S.D.) are obtained from eight individual experiments (chimeric
constructs), four experiments (p0Luc), and five experiments
(pSVLuc).
We made five different
AT receptor-luciferase expression vectors and transfected
these into R3T3 cells. As shown in Fig. 2, A and B, deletion of -453/-225 fragment enhanced
luciferase activity 4-fold in growing R3T3 cells when compared to other
constructs. This result suggests that the sequence between -453
and -225 may contain functional negative regulatory elements for
AT
receptor gene expression during cell growth. However, in
confluent R3T3 cells, we could not observe any significant differences
in the expressed luciferase activities. Interestingly, we found in this
region the several putative consensus sequences such as two repeats
(5`-AAGAGAAAGAGA-3`) of IRF binding sequence (30) at the
position -282 bp and this hexamer motif (5`-AAATGA3-`) at the
position -258 bp (Fig. 1B). We also found protein
kinase C-responsive element (AP1) (31) at position -340
bp and octamer binding sequence (5`-ATTCAAAAT-3`) for POU domain family
of transcriptional factors (32) at position -394 bp (Fig. 1B). In addition, we also demonstrated that
luciferase activity caused by p(-224/+52)Luc transfection in
growing R3T3 cells was still lower (approximately 80%, p <
0.05) than that of confluent R3T3 cells transfected by same expression
vector construct (Fig. 2, A and B). This
result suggests the existence of an additional mechanism for positive
regulation of the AT
receptor gene expression in these
cells from growing to quiescent state. Alternatively, there exists
another weak negative regulatory element between the positions
-224 and +52.
We focused on the molecular mechanism of
negative regulation of AT receptor expression during R3T3
cell growth by the putative negative regulatory elements located
between -453 and -225 bp. To examine which sequences are
the specific cis DNA elements involved in the negative
regulation, we performed DNase footprinting analysis. We observed two
distinct protected regions as shown in Fig. 3, A and B, and more clear protected regions were obtained with a
higher concentration of nuclear extracts, suggesting that these sites
are specifically protected by nuclear proteins. However, there were no
significant differences in protected regions and their intensities
between growing and confluent cells. Interestingly, these regions
contain putative octamer binding motif and IRF binding sequence,
respectively.
To examine these protected regions, which are in fact
involved in the negative regulation of AT receptor
expression in growing R3T3 cells, we made double-stranded
oligonucleotides corresponding to these protected sequences (see
``Experimental Procedures'') and delivered these
oligonucleotides to R3T3 cells to act as ``decoy.'' This
assay is based on the competition between the cis elements
present in an endogenous target gene and exogenously added
oligonucleotides corresponding to the cis sequence. This
method seems advantageous compared to performing the further deletion
of the promoter region and examining the effects of putative sequences
on luciferase or CAT expression because we can observe the direct
effects of candidate cis elements on the AT
receptor binding and expression. To deliver the oligonucleotides
efficiently into R3T3 cells, we employed the HVJ-liposome delivery
method(26, 27) . By this transfer method, we observed
that nuclei of more than 90% of the cells were stained with transfected
fluorescein isothiocyanate-labeled oligonucleotides 1 h after
transfection, and the stained cells continued up to at least 3 days.
Three days after transfection of oligonucleotides, AT receptor binding was measured. As shown in Fig. 3, C and D, octamer binding oligonucleotide transfection did
not affect AT
receptor binding both in growing and
confluent R3T3 cells, suggesting that this sequence alone may not play
an important role in AT
receptor expression in R3T3 cells.
On the other hand, IRF binding oligonucleotide treatment increased the
AT
receptor binding in growing R3T3 cells but not in
confluent cells. However, mutant IRF binding decoy oligonucleotide
treatment did not affect the AT
receptor binding,
supporting the notion that this effect is specific for IRF binding. We
also measured AT
receptor mRNA levels at 24 h after
oligonucleotide treatment and observed that IRF binding decoy
oligonucleotide increased AT
receptor mRNA in growing R3T3
cells only, whereas octamer binding oligonucleotide did not affect
AT
receptor mRNA level, supporting the contention that the
changes of AT
receptor expression are transcriptionally
regulated (Fig. 3E). These results indicate that IRF
binding sequence may play a critical role in the negative regulation of
AT
receptor in growing R3T3 cells.
Next, we examined
whether specific nuclear transcriptional proteins recognize this
sequence. Gel mobility shift assay using IRF binding oligonucleotide
showed a specific retarded band both in growing and confluent R3T3
cells (Fig. 4A). This specific binding was abolished by
the addition of C1 oligomer(25) , which is known to bind
specifically to IRFs. Furthermore, as shown in Fig. 4B,
we demonstrated the specific binding to P-labeled C1
oligomer that was competed by the IRF binding oligonucleotide,
supporting that IRFs recognize this region. IRF-1 and IRF-2 are
structurally related, particularly in their amino-terminal regions,
which confer DNA binding specificity, and they recognize the same DNA
sequence elements(12, 14) . IRF-1 and IRF-2 bind to
the promoters of IFN-
, IFN-
, and several IFN-inducible genes
and modulate these gene
expressions(12, 13, 14, 15, 16, 17) .
Gene transfection studies have demonstrated that these two factors are
mutually antagonistic; IRF-1 activates transcription whereas IRF-2
inhibits it(14, 15) .
Figure 4:
IRF binding to AT receptor
promoter and expression in R3T3 cells. A, gel shift analysis
of IRF to
P-labeled IRF binding oligonucleotide with the
nuclear extracts prepared from R3T3 cells in growing (lanes2-5, 5, 10, 15, and 15 µg of protein,
respectively) and confluent states (lanes6-9,
5, 10, 15, and 15 µg of protein, respectively). Lanes5 and 9 were carried out with 100-fold molar
excess of C1 oligomer (AAGTGA)
(25) as competitor. Lane1, without nuclear extract. B, gel
shift analysis of IRF to
P-labeled C1 oligomer with the
nuclear extracts prepared from R3T3 cells in growing (lanes2-4, 5, 10, and 10 µg of protein, respectively)
and confluent states (lanes5-7, 5, 10, and 10
µg of protein, respectively). Lanes4 and 7 were carried out with 100-fold molar excess of IRF binding
oligonucleotide as competitor. Lane1, without
nuclear extract. C, RT-PCR analysis of IRF-1 (leftpanel), IRF-2 (middlepanel), and
-actin expressions (rightpanel) in R3T3 cells.
Total RNA (lanes1 and 4, 0.12 µg; lanes2 and 4, 0.24 µg; lanes3 and 5, 0.36 µg) prepared from growing (lanes1-3) and confluent (lanes4-6) R3T3 cells was subjected to RT-PCR. The lanes on the rightsides show HaeIII-digested
X marker. D, gel shift analysis
of IRF to
P-labeled IRF binding oligonucleotide with the
nuclear extracts (15 µg) prepared from R3T3 cells in growing and
confluent states with 2 µg of IRF-1 or IRF-2 antibody (Santa Cruz
Biotechnology). Arrow indicates IRF
binding.
Accordingly, we hypothesized
that IRF-2 may act as silencer for AT receptor expression
in R3T3 cells, and IRF-1 enhances AT
receptor expression by
interacting with the AT
receptor promoter region. To test
this hypothesis, we transfected antisense oligonucleotides for IRF-1
and IRF-2 into R3T3 cells and observed that IRF-2 antisense enhanced
the AT
receptor binding both in growing and confluent R3T3
cells (Fig. 5, C and D), and sense
oligonucleotide for IRF-2 did not influence the AT
receptor
binding. On the other hand, IRF-1 antisense oligonucleotide attenuated
the AT
receptor binding in confluent R3T3 cells but had no
effect in growing cells (Fig. 5, A and B).
These changes in AT
receptor binding levels in response to
antisense treatment were accompanied by changes in the AT
receptor mRNA levels (Fig. 5E), supporting
further that IRF-1 and IRF-2 act as transcription regulators for
AT
receptor expression. These results suggest that IRF-2
binding decreases the AT
receptor level both in growing and
confluent R3T3 cells, whereas IRF-1 binding counteracts IRF-2 effect by
competing the binding of IRF-2 in the confluent R3T3 cells. Consistent
with this result, we observed that IRF-1 mRNA expression was more
abundant in confluent cells than growing cells, whereas IRF-2
expression did not change with R3T3 cell growth (Fig. 4C). We also demonstrated that IRF binding in
growing cells was mainly with IRF-2, whereas the IRF binding in
confluent cells was with both IRF-1 and IRF-2 (Fig. 4D). These data suggest that the ratio of IRF-1
and IRF-2 may determine the AT
receptor expression
associated with the growth of R3T3 cells. In addition to IRF family
proteins such as IRF-1 and IRF-2, elucidation of the roles of other
transcription factors such as interferon consensus sequence binding
protein (20) may provide further understanding of the mechanism
of growth-regulated AT
receptor expression.
Figure 5:
AT receptor binding (A-D) and expression (E) by the treatment of
antisense oligonucleotides for IRF-1 and IRF-2. Antisense
oligonucleotides for IRF-1 (A and B) and IRF-2 (C and D) were transfected into R3T3 cells in growing state (A and C) and confluent state (B, D). The
AT
receptor binding in untreated cells is taken as 100%,
respectively. The values were expressed as mean ± S.D. obtained
from eight different cell culture wells. E, total RNA was
prepared from cultured R3T3 cells 24 h after oligonucleotide treatment.
RNA (20 µg/lane) was separated by electrophoresis, and
hybridization was carried with a probe for the AT
receptor
or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a
control. The result is a representative of data obtained in three
different experiments.
IRF-1
manifests antiproliferative properties(33, 34) .
Taniguchi and colleagues (33) 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. Furthermore, overexpression of the repressor
IRF-2 in these cells leads to cell transformation and increased
tumorgenicity, and this phenotype can be reverted by the concomitant
overexpression of IRF-1(35) . IRF-2 is also reported to be more
abundant in growing cells than IRF-1, but after stimulation by IFN or
viruses, the amount of IRF-1 increases relative to IRF-2(36) .
Interestingly, in addition to the IFN system, IRF binding consensus
element was recently identified in inducible nitric oxide synthase gene
promoter region and IRF-1 is essential for inducible nitric oxide
synthase activation in murine macrophages(19) . In this paper,
we report that IRFs may regulate the AT2 receptor expression during
cell growth and that AT receptor is one of the target genes
of IRF system.
Janiak et al.(37) has shown that
CGP42112A, an AT receptor ligand suspected to be an
AT
receptor agonist, attenuates neointimal development
after vascular injury. We also observed that overexpression of AT
receptor in balloon-injured rat carotid artery attenuates the
neointimal formation(38) . Daud et al.(39) reported that the granulosa cells of the atretic
ovarian follicles, with an event dependent on apoptosis, express high
levels of Ang II receptors, and Pucell et al.(6) also
reported that the follicular granulosa cell Ang II receptors are the
AT
receptor. Our preliminary results demonstrated that Ang
II enhanced apoptotic changes in confluent R3T3 cells(40) .
These results suggest that the AT
receptor exerts
antigrowth effects on cell growth and may induce apoptosis in some
cells. Tanaka et al.(18) recently reported that IRF-1
may be a critical determinant of oncogene-induced cell transformation
or apoptosis in mouse embryonic fibroblast. Taken together, these
results provide us the hypothesis that IRFs regulate cell growth and/or
differentiation by modulating AT
receptor expression,
thereby supporting a role for this receptor in these processes. Our
results also will give further insights into the role of IRF-1/IRF-2 in
the regulation of cell growth.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].