| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
INSERM-INRA U 418, IFREL, and Université Claude Bernard, Hôpital Debrousse, 69322 Lyon Cedex 05, France
Address all correspondence and requests for reprints to: J. Yuan Li, INSERM-INRA U418, Hôpital Debrousse, 29 rue Soeur Bouvier, 69322 Lyon Cedex 05, France. E-mail: liyuan{at}lyon151.inserm.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Studies on the regulation of the AT2 receptor have used either cells expressing both AT1 and AT2 (9, 10) or cells expressing exclusively AT2 receptors (11, 12, 13, 14, 15, 16, 17, 18, 19). In the first group of cells, AngII is one of the major negative regulators of AT2 binding sites and messenger RNA (mRNA), and these effects are mediated through AT1 receptors (10). In contrast, in the second group of cells, the main negative regulators of AT2 are serum and several growth factors (12, 13, 14, 15, 16, 17, 18). Contradictory results have been reported concerning the effects of AngII on AT2 receptors: lack of effects (20), reduction (11), or increase of AT2 binding sites (12, 13, 15, 17). These discrepancies on the effects of AngII on its own receptor might be related to differences in the cell type used. However, in most cases, the mechanisms (transcriptional or posttranscriptional) by which these different factors regulate AT2 receptors have not been investigated.
In the present work, using R3T3 cells at early passages, we have investigated the mechanism by which AngII and three growth factors [insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor ß1 (TGFß1)] regulate AT2 expression. We demonstrate that AngII acts mainly at the translational level, whereas the stimulatory (IGF-1) or inhibitory (bFGF and TGFß) effects of these growth factors are exerted at the transcriptional level.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell culture
Cells were grown in DMEM/F12 supplemented with antibiotics and
10% FCS. For the experiment described below, cells were plated at a
density of 5–7 x 104 cells/cm2 and
cultured in the same medium. After 4–5 days, time required for
confluency, the medium was replaced by DMEM/F12 supplemented with
antibiotics and 0.1% BSA (BSA) for 2 additional days. Then, cells were
incubated for 2 days in the same medium without (control) or with AngII
(10-7 M), IGF-1 (50 ng/ml), bFGF (10 ng/ml),
or TGFß1 (2 ng/ml). These concentrations were chosen because, in
preliminary experiments, they were found to produce maximal responses.
The medium was renewed daily. At the end of each experiment, cell
numbers for each experimental condition were counted in triplicate in a
Coulter counter (model ZBI, Coulter Electronics, Hialeah, FL).
Radioligand binding assay
CGP42112 was radiolabeled by the Iodogen method with
125I and then purified by HPLC using a C18 Bondapak column
(Millipore Corp., Guyancourt, France) eluted with a
10–60% linear acetonitrile gradient in 0.1% trifluoroacetic acid.
The specific activity of the isolated monoiodinated peptide, as
measured by self-displacement in a radioreceptor assay, was 1800–2000
Ci/mmol.
The receptor assay was carried out using 24-well plates containing
0.3–0.6 x 106 cells/well. Binding was carried out
for 2 h at 37 C (equilibrium conditions) in 0.5 ml binding medium
(DMEM/F12, 0.5% BSA, 0.1% bacitracin, and 10 mM HEPES, pH
7.4) containing either the radioactive tracer (0.10 nM) and
varying concentrations of unlabeled ligand or 1 nM of the
radio-tracer. Nonspecific binding was evaluated in the presence of
10-6 M AngII or 10-7
M CGP42112, with identical results. At the end of the
incubation, the medium was removed, and the cells were washed three
times with 0.9% NaCl 0.5% BSA and then dissolved in 0.5 M
NaOH 0.4% sodium deoxycholate. The radioactivity of the binding assays
was measured in a 
-counter and corrected by the number of cells for
each experimental condition.
Autoradiography
After binding, cells were prefixed in 0.5% (vol/vol)
glutaraldehyde in PBS for 4 min at room temperature. This procedure
reduced total binding by only 2–4%. Then, cells were washed four
times, dried, and fixed in 4% (wt/vol) paraformaldehyde (PFA) in PBS
for 10 min at room temperature. After dehydration, cells were coated
with LM1 photographic emulsion (Amersham Pharmacia Biotech, Les Ulis, France), air dried, then exposed for 1 week
at 4 C. Cells were stained with hematoxylin-eosin before being examined
with a Carl Zeiss microscope and photographed.
RNA isolation and Northern blot analysis
Total RNA was isolated from cells by the method of Chomczynski
and Sacchi (22). For Northern blot analysis, total cytoplasmic RNA
(15–40 µg) was subjected to electrophoresis through 1% agarose gels
containing 8% formaldehyde. The RNA transfer to hybond-N membranes,
the prehybridization, the hybridization using labeled AT2
cDNA (21), and washings were performed as previously described (14).
Autoradiograms were obtained after 2–6 days exposure, at -70 C, to
Hyperfilm MP (Amersham Pharmacia Biotech) with
intensifying screens. Autoradiograms and 28S RNA ethidium bromide
fluorescence photographs were submitted to densitometry scanning using
an image analyzer, Samba 200S (Alcatel, Grenoble, France).
AT2 mRNA stability
To measure the half-life of AT2 mRNA, cells (at the
end of 48-h treatments) were incubated with the same medium, containing
actinomycin D (5 µg/ml). After 2 h, the incorporation of
[3H]uridine into trichloroacetic acid-precipitable
material was less than 5% of that of nontreated cells. At the
indicated times, AT2 mRNA was measured by Northern
blot.
In situ hybridization
The AT2-receptor template was amplified from
the mouse AT2 receptor cDNA in a PCR reaction consisting of
a 5-min denaturation step, at 94 C, followed by 35 amplification cycles
(94 C for 1 min; 65 C for 1 min, and 72 C for 1 min) and a 10-min
elongation step, at 72 C, in the presence of the following primers:
sense primer,
5'-CAGAGATGC-ATTAACCCTCACTAAAGGGAGA/CCGGGATGTCAGAACCATTGAA-3'
(the consensus T3 sequence is shown in bold
letters, preceded by a 9-bp leader sequence in italics, and
followed after the slash (/) by the gene-specific sequence 543–564)
and antisense primer,
5'-CCAAGCTTCTAATACGACTCACTATAGGGAGA/GC-CTTGGAGCCAAGTAATGGGAAC-3'
(the consensus T7 sequence is shown in bold
letters, preceded by a 9-bp leader sequence in italics, and
followed after the slash (/) by the gene-specific sequence 1026–1003).
The resulting PCR products were purified using QIA quick-spin columns
from QIAGEN (Courtaboeuf, France). 33P
labeling during transcription of the complementary RNA (cRNA) probe was
performed essentially as described by Logel (23).
In situ hybridization, using cRNA probes, was carried out as described by Simmons (24), with some modifications. Briefly, after culture, cells were scraped, centrifuged on microscope glass-slides, air dried, and fixed in 4% (wt/vol) PFA in PBS for 10 min at room temperature. After dehydration, cells were stored at -20 C until use. Cells were rehydrated and permeabilized with 1 µg/ml proteinase K (Roche Molecular Biochemicals) for 15 min at 37 C. Then, they were incubated in 4% PFA for 5 min, dehydrated, and air dried. The cell spots were hybridized with either 33P-labeled antisense RNA probe or sense RNA probe in a solution containing 50% (vol/vol) deionized formamide, 300 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 x Denhardt’s solution, and 10% dextran sulfate. Hybridization solution (20 µl) containing 2 x 106 cpm was placed over each cell spot and covered with a 20 x 20-mm chloroform-washed coverslip. Cells were hybridized by incubation overnight at 55 C in a humidified chamber. After hybridization, the slides were treated with ribonuclease A (20 µg/ml) at 37 C for 15 min and washed in SSC buffers with decreasing salt concentrations; and the final posthybridization wash was in 0.1 x SSC at 65 C for 30 min, twice. Slides were quickly dehydrated in ethanol and air dried. Slides were then dipped in LM1 emulsion diluted 2:3 with water (vol/vol) (Amersham Pharmacia Biotech) at 42 C, exposed for 1 or 2 weeks at 4 C, developed in D-19 developer (Eastman Kodak Co., Eubonne, France), and fixed in Unifix (Eastman Kodak). Then cells were counterstained with hematoxylin-eosin before being examined and photographed. Sense RNA probe was used as a control for nonspecific binding.
Nuclear run-on transcription
This technique, which is capable of detecting changes in the
transcription rate of genes, was performed as previously described
(25). Nuclei (3–4 x 107) isolated from R3T3 cells,
treated with or without growth factors, were incubated for 30 min at 26
C in a reaction buffer containing ATP, cytidine 5'-triphosphate, GTP,
and [
-32P]uridine 5'-triphosphate (Amersham Pharmacia Biotech). 32P-labeled RNA was extracted
and hybridized (1–1.5 x 106 cpm/ml hybridization
buffer) for 60 h at 65 C with AT2 and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) cDNAs immobilized on nitrocellulose
membranes. After hybridization, the filters were washed four times (15
min each) at 65 C in 2 x NaCl/Cit (NaCl/Cit: 160 mM
NaCl, 15 mM sodium citrate, pH 7.4), 0.1% SDS, then twice
(30 min each) at 65 C in 0.2 x NaCl/Cit, 0.1% SDS. The filters
were incubated at 37 C in 2 x NaCl/Cit at 37 C for 1–2 h.
Filters were exposed to Hyperfilm M. P. at -70 C for 3–14 days.
The rates of gene transcription were determined by densitometric
analysis of blots and were normalized by expressing them as ratios to
the GAPDH signals.
Isolation of polysomes and mRNA preparation
At the end of the treatments indicated above, cells were rinsed
and collected in ice-cold PBS (pH 7.4) with 10 µg/ml cycloheximide
before the cells were pelleted (100 x g for 10 min).
The cell pellet was resuspended in 500 µl lysis buffer [20
mM Tris/HCl (pH 8.0), 1.5 mM MgCl2,
140 mM KCl, 0.5 mM dithiothreitol, 0.2
mM cycloheximide, 0.5% Nonidet P-40, 0.1 mM
phenylmethylsufonyl fluoride, 2 µg/ml leupeptin, 8 µg/ml aprotinin,
and 1000 U/ml RNasin] and centrifuged at 10,000 x g
for 10 min at 4 C. The supernatant was layered over a 12-ml linear
20–47% sucrose gradient containing 20 mM Tris/HCl (pH
8.0), 140 mM KCl with 5 mM MgCl2 or
10 mM EDTA and was centrifuged, for 2 h and 15 min at
150,000 x g, with a Beckman Coulter, Inc.
SW41 rotor. The gradients were emptied from the bottom, their
absorbance was read at 260 nm with an UV detector, and they were
sampled (0.5 ml). To each sample were added 5 µl of 20% SDS and 10
µl of 10 ml/ml proteinase K; then they were incubated at 37 C for 15
min and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The
RNAs corresponding to two successive fractions were pooled and were
precipitated overnight with an equal volume of 2-propanol. Then, the
samples were subjected to slot blot. After denaturation, RNAs were
transferred directly to Hybond-N nylon membranes using a multiwell
filtration manifold (Life Technologies, Inc.).
Membranes with bound RNA were baked at 80 C for 2 h and irradiated
by UV. Hybridization with the 32P-labeled cDNA probes and
analysis of the blots were carried out as described above for Northern
blot.
Statistical analysis
All data are presented as mean ± SEM of the
number of experiments indicated in the legends of the figures, except
for Fig. 5
. Comparisons between control and treated cells were carried
out by multifactorial ANOVA, taking into account the different
independent cultures and the experimental conditions. P
< 0.05 was considered as significant. The data from the distribution
of AngII type 2 (AT2) mRNA in the sucrose gradient (see
Fig. 7) were analyzed by two-way ANOVA. For this analysis, the
fractions were pooled into heavy polysomal fractions (fractions
1–6), medium-light polysomal fractions (fractions 7–11), and free
mRNA fractions (fractions 17–20).
|
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). In
contrast, IGF-1 increased the two parameters, whereas bFGF, TGFß, and
FCS reduced both.
|
). AT2 agonist CGP 42112 had
effects similar to those of AngII on both AT2 binding sites
and mRNA levels, and no additive effects were found in cells treated
with both compounds. Moreover, AngII did not modify the stimulatory
(IGF-1) or inhibitory (bFGF, TGFß, or FCS) actions of these factors
on AT2 mRNA levels. In contrast, AngII significantly
increased AT2 receptor in cells treated with any of the
growth factors. This effect of AngII was more striking in cells treated
with IGF-1, in which the combined effect of the two factors was more
than additive.
|
). At maximal
effective concentrations of AngII (10-7 M), a
stimulatory effect was observed within 12 h and was maximal at
36 h. The threshold and the maximal responses were observed at
10-9 M and 10-7 M,
respectively.
|
). Both AngII and IGF-1 increased the
binding of [125I]CGP 42112, but the effects were again
less pronounced than that produced by AngII + IGF-1. However, because
of the very high density of the signal, it was not possible to evaluate
the number of grains per cell. In contrast, bFGF markedly reduced the
binding. Analysis of the in situ hybridization
autoradiograms revealed that only IGF-1, and IGF-1 + AngII, increased
significantly the number of grains per cell (control = 27 ±
12, AngII = 37 ± 15, IGF-1 = 115 ± 22, IGF-1 +
AngII = 106 ± 19, bFGF = 3 ± 1.5, n = 50–60
cells for each condition). These results suggest that, in R3T3 cells,
AngII and growth factors regulate AT2 receptor by different
mechanisms.
|
), indicating that
none of these factors modified the stability of AT2 mRNA.
In contrast, TGFß1 and bFGF reduced by 80 and 92%, respectively, the
rate of AT2 transcription, whereas IGF-1 increased it by
250% (Fig. 6). These effects, similar to
those seen for AT2 mRNA levels (Fig. 1), suggest that the
main mechanism by which these growth factors regulate AT2
mRNA is at the level of transcription. Moreover, AngII alone or
together with IGF-1 had no significant effect on AT2 gene
transcription.
|
RNA extracts from two successive fractions (1 with 2, 3 with 4, and so
on) were pooled and subjected to slot blots. In control cells,
AT2 mRNA spread throughout the polysomal fractions, with a
peak in the medium-size polysomes and another peak in the top of the
gradients (Fig. 7). In cells treated with
IGF-1, AT2 transcripts also spread throughout the polysomal
fractions, with a plateau in the medium light-size polysomes and one
small peak in the free mRNA fraction. In contrast, in cells treated
with AngII, and those treated with AngII + IGF-1, AT2 mRNA
was located mainly in the heavy polysomal fractions; and the transcript
almost disappeared in the free mRNA fractions. In contrast, the
distribution of GAPDH mRNA was not modified by any treatment (Fig. 7).
Statistical analysis (ANOVA) of the mRNA distribution confirmed that
AngII alone or together with IGF-1 significantly increased
(P < 0.006) AT2 mRNA in the heavy
polysomal fractions (fractions 1–6) and decreased (P
< 0.002) AT2 mRNA in free mRNA fractions (fractions
17–20). In contrast, IGF-1 increased (P < 0.001)
AT2 mRNA in medium-light fractions (fractions 7–11).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Regulation of AT2 receptors by growth factors has been reported in several cell types, including PC12W (14, 17, 18), rat vascular smooth muscle cells (29, 30), rat neurons (31), and R3T3 cells (12, 14, 16, 18). By using several approaches, binding assays, autoradiography, Northern blot, and in situ hybridization, our results clearly demonstrated that, in R3T3 cells, IGF-1 increased, whereas bFGF and TGFß1 decreased both AT2 binding sites and mRNA levels. The main mechanism by which these growth factors regulate AT2 expression is at the transcriptional level, because none of these factors modified significantly AT2 mRNA half-life, whereas the rate of transcription of the AT2 gene, evaluated by run-on assay, was increased by IGF-1 and decreased by bFGF and TGFß1.
The promoter of the mouse AT2 receptor contains several putative consensus sequences, such as AP-1, C/EBP, an insulin response sequence, and an interferon regulatory factor (IRF) binding motif (27, 30, 32). Although not yet proven, the AP-1 site might be involved in the negative regulation of AT2 expression induced by phorbol esters and probably by bFGF. Similarly, the C/EBP might be involved in the up-regulation of AT2 expression induced by interleukin 1ß (28). On the other hand, convincing evidence has indicated that AT2 receptor is regulated by the IRF system. In R3T3 cells, serum starvation increased the ratio of IRF-1 to IRF-2 (IRF-1 stimulates, whereas IRF-2 inhibits, AT2 gene transcription), mediated the up-regulation of AT2, and induced apoptosis (19, 27, 28). However, it is still unknown whether these changes in the ratio of IRF-1 to IRF-2 are also involved in the opposite effects of IGF-1 and bFGF or TGFß1 on AT2 expression and whether the up-regulation of AT2 is always associated with increased apoptosis. The latter seems not to be the case, because IGF-1, which stimulates both cell multiplication (19) and AT2 expression (present results), is antiapoptotic in R3T3 cells (our own unpublished results).
Regulation of AT2 receptor by AngII has been studied in several cell models, and some of the results are conflicting. In cells expressing both AT1 and AT2 receptors, AngII either down-regulated AT2 binding sites (9, 10) and mRNA (10) or increased AT2 protein (15); and in both cases, the effects were mediated through AT1 receptors. In PC12W cells and cultured bovine thecal cells, which express only AT2 receptors, AngII had no effects on AT2 receptors (10, 30); whereas in rat granulosa cells, which also express AT2 receptors, AngII reduced the number of AT2 binding sites (11). In contrast, in mouse embryo fibroblasts (12) and in R3T3 cells (12, 16), AngII increased AT2 binding sites (12) and/or mRNA levels (13, 16).
The present findings confirm previous studies showing the positive effects of AngII on AT2 receptors and provide data to understand the mechanism by which AngII up-regulates these receptors. First, the effects were mediated by AT2 receptors, because the effects of AngII were mimicked by the AT2 agonist CGP42112 and blocked by the AT2 antagonist PD123177, although this compound alone had no effect. These results are in contradiction with those reported previously (12) showing that the AT1 antagonist (Sar1, Ala8-AngII) and AT2 antagonists (PD123319 and PD123177) enhanced AT2 receptor number. The reasons for this discrepancy are unknown. Second, AngII and/or CGP42112 did not modify AT2 mRNA levels, as demonstrated by Northern blot and in situ hybridization. Third, AngII was unable to modify either AT2 mRNA half-life or AT2 gene transcription. Taken together, these results suggest that AngII acts at the translational level.
A means of studying the translational efficiency of an mRNA species is to evaluate, in cell extracts run through a sucrose density gradient, its relative distribution in polysomal fractions (i.e. the mRNAs engaged into translation) and in the subpolysomal fractions (i.e. the free mRNAs) (33, 34, 35, 36). Using this approach, we found a marked increase of AT2 mRNA in heavy polysomal fractions and a decrease in free mRNA fractions in cells treated with AngII or AngII plus IGF-1, when compared with control and IGF-1-treated cells. Our results also indicated that IGF-1 slightly increased the rate of translation. In contrast, the distribution of GAPDH mRNA was not modified by any treatment. Because AngII modified neither AT2 gene expression nor AT2 mRNA stability, the mechanism by which this peptide enhanced the rate of AT2 mRNA translation might be related to some factor involved in either the shift of AT2 mRNA from messenger ribonucleoprotein particles into polysomes and/or in the initiation or elongation of AT2 mRNA (37, 38). Further studies are required to elucidate this point.
In conclusion, our study has demonstrated the complexity of AT2 receptor regulation. Thus, the main role of growth factors is to regulate, positively or negatively, the rate of transcription of the AT2 gene, whereas AngII only increases the rate of translation of AT2 mRNA.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received February 24, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  V. M. Portela, P. B. D. Goncalves, A. M. Veiga, E. Nicola, J. Buratini Jr., and C. A. Price Regulation of Angiotensin Type 2 Receptor in Bovine Granulosa Cells Endocrinology, October 1, 2008; 149(10): 5004 - 5011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Esteban, O. Lorenzo, M. Ruperez, Y. Suzuki, S. Mezzano, J. Blanco, M. Kretzler, T. Sugaya, J. Egido, and M. Ruiz-Ortega Angiotensin II, via AT1 and AT2 Receptors and NF-{kappa}B Pathway, Regulates the Inflammatory Response in Unilateral Ureteral Obstruction J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1514 - 1529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Adamy, P. Oliviero, S. Eddahibi, L. Rappaport, J.-L. Samuel, E. Teiger, and C. Chassagne Cardiac modulations of ANG II receptor expression in rats with hypoxic pulmonary hypertension Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H733 - H740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stoll, A. W.A. Hahn, U. Jonas, Y. Zhao, B. Schieffer, J. W. Fischer, and T. Unger Identification of a Zinc Finger Homoeodomain Enhancer Protein After AT2 Receptor Stimulation by Differential mRNA Display Arterioscler Thromb Vasc Biol, February 1, 2002; 22(2): 231 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Uhlenius, O. Vuolteenaho, and I. Tikkanen Renin-angiotensin blockade improves renal cGMP production via non-AT2-receptor mediated mechanisms in hypertension-induced by chronic NOS inhibition in rat Journal of Renin-Angiotensin-Aldosterone System, December 1, 2001; 2(4): 233 - 239. [Abstract] [PDF]
|
|
|
|
 |
|
 S. H. Park and H. J. Han The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells Am J Physiol Renal Physiol, February 1, 2002; 282(2): F228 - F237. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
