1 Institut National de la
Santé et de la Recherche Médicale, Angiotensin II
(ANG II) has long been known for its pressor and growth-promoting
effects, which are both mediated by the
AT1 receptor. By contrast, the
AT2 receptor has recently been
reported to mediate inhibition of proliferation through as yet
undefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks the
cells in G1 phase. Consistent with
this, ANG II inhibits cyclin D1 expression and cyclin
D1-associated kinase activity. The
antimitogenic effect of ANG II is partly mimicked by the
AT2-selective agonist CGP-42112.
It is also blocked partly and in an additive fashion by the
AT1- and
AT2-selective antagonists losartan
and PD-123319, indicating the contribution of both receptor subtypes to
this response. AT1-dependent
antiproliferation is selectively blocked by the cyclooxygenase
inhibitor indomethacin and restored by prostaglandin E2, whereas
AT2-receptor-mediated inhibition
of growth is suppressed by the tyrosine phosphatase inhibitors
orthovanadate and bpV(pic). Both pathways are, however,
pertussis toxin sensitive. We hypothesize that, in fasciculata
cells, the AT1 receptor inhibits
bFGF-induced proliferation by stimulating prostaglandin synthesis,
whereas the AT2 receptor mediates
its effect through a pathway that requires protein tyrosine phosphatase
activation.
cyclin D; cyclin-dependent kinases; protein tyrosine phosphatase; G
proteins; prostaglandin; angiotensin II; angiotensin receptors; basic
fibroblast growth factor
THE VASOACTIVE OCTAPEPTIDE angiotensin II (ANG II)
interacts with two subtypes of receptors,
AT1 and
AT2, which have recently been
cloned (8). Although they both belong to the seven-transmembrane-domain receptor superfamily, they only share 34% identity and differ in their
signaling pathways. The
AT1 receptor, which is expressed in virtually all known target tissues of ANG II, mediates the blood
pressure- and body fluid-regulatory actions of the peptide through
G-protein-coupled pathways that have been extensively studied (2, 8).
The signaling mechanisms and physiological functions of the
AT2 receptor are much less well
understood. We have previously reported data indicating that this
receptor mediates inhibition of the atrial naturetic peptide (ANP)
receptor guanylate cyclase activity (4) and modulates T-type calcium
currents (6). We have also shown that this receptor
stimulates a protein tyrosine phosphatase (PTP) activity (5), which is
involved in both biological responses described above (4, 6).
Activation of a PTP in AT2
receptor signaling has been confirmed by others (21) and has recently
been proposed to be involved in
AT2 receptor-mediated apoptosis
(37).
The importance of protein tyrosine phosphorylation and
dephosphorylation in the signaling pathways of growth factors has
prompted us and others to investigate the potential role of the
AT2 receptor in the regulation of
cell proliferation. We recently reported that this receptor mediates
the inhibition of growth factor-induced endothelial (32) and PC-12 cell
(20) proliferation, an effect that was confirmed by others in vascular
smooth muscle cells transfected with the
AT2 receptor (22). The mechanisms
involved in this response are, however, still unknown.
By contrast and apart from its role in mediating the cardiovascular
actions of ANG II, the AT1
receptor has also been reported to mediate growth-promoting effects in
a variety of cells and tissues, and ANG II is generally considered to
be a major stimulator of neointimal proliferation (16). The
growth-regulatory properties and mechanisms of ANG II thus appear
complex and dependent on the receptor subtype(s) and signaling pathways
expressed by the target cell.
The aim of the present study was to assess the growth-regulatory
actions of ANG II on cells expressing functional
AT1 and AT2 receptors and to investigate
their respective contribution to this response.
For this purpose, we used bovine adrenal fasciculata cells (BAC) in
their differentiated phenotype, as defined by their ability to produce
cortisol in response to adrenocorticotropic hormone (ACTH) and ANG II
(11, 26). The adrenal gland is one of the major target organs of ANG
II, in which it not only regulates steroid production but also acts as
a major growth factor (9, 15). These cells express
AT1 and
AT2 receptors (23), both of which
mediate steroidogenesis, apparently through different pathways (10).
The AT2 receptor also mediates
inhibition of ANP-dependent guanylate cyclase in these cells (10).
In this study, we were unable to detect any significant mitogenic
effect of ANG II and, therefore, investigated the ability of this
peptide to modulate the growth-promoting effects of basic fibroblast
growth factor (bFGF), known to be mitogenic in these cells. We show
here that ANG II inhibits bFGF-stimulated proliferation but that, in
contrast to the data reported that were obtained by using endothelial
and vascular smooth muscle cells (22, 32), in BAC this effect is
mediated by both AT1 and
AT2 receptors. This
antiproliferative response is due to blockade of the cells in the
G1 phase of the cycle. Analysis of
the mechanisms involved suggests that the
AT1 receptor mediates this effect
through the stimulation of prostaglandin synthesis, whereas the
AT2-receptor signaling pathway
involves activation of a PTP. Both pathways, however, lead to reduced
cyclin D1 expression and to inhibition of cyclin
D1-dependent kinase activity.
These results stress the dual action of ANG II on cell proliferation
and indicate that the growth response to this peptide does not depend
only on the receptor subtype that is expressed but rather depends on
the signaling pathways it is coupled to and that vary according to the
cell type and its state of differentiation.
Cell isolation and culture.
BAC were prepared as described previously (11) and were grown during 24 h in Ham's F-12 medium supplemented with 10% horse serum and 2.5%
fetal calf serum. Subsequently, they were starved for 72 h in Ham's
F-12 with 0.1% bovine serum albumin (BSA) to achieve quiescence.
Radioligand binding assays.
Competition binding experiments were performed on membrane particulate
as described previously (3), using either
125I-labeled
[Sar1,Ile8]ANG
II (0.25 nM) or 125I-labeled
CGP-42112 (0.25 nM) as a tracer and ANG II, losartan, and CGP-42112 as
competing ligands. Nonspecific binding was determined in the presence
of 1 µM ANG II. Degradation of
125I-[Sar1,Ile8]ANG
II and 125I-CGP-42112 as measured
by thin-layer chromatography was <5%. Data were analyzed by using
the nonlinear regression program LIGAND.
DNA synthesis and cell proliferation assays.
DNA synthesis was assessed in triplicate wells by incorporation of
[3H]thymidine. BAC
were seeded in 22-mm wells at a density of
105 cells/well. Quiescent cells
were incubated in fresh Ham's F-12 containing 0.1% BSA with the
various effectors (bFGF, ANG II, losartan, valsartan, CGP-42112,
PD-123319) for the indicated periods of time, and 0.25 µCi
[3H]thymidine (87 Ci/mmol) was added to each well 3 h before the end of the
incubation time. Radioactivity incorporated into
trichloracetic acid-insoluble material was measured by scintillation
counting.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cell-cycle analysis by flow cytometry. BAC were seeded in 10-cm dishes at a density of 3 × 106 cells/dish. After 30 h of incubation with the effectors, 5-bromo-2'-deoxyuridine (BrdU; 5 mM) was added and the incubation was pursued for 20 min. The cells were washed, trypsinized, and fixed in 70% ethanol. After permeabilization with Tween 20 (0.5%), the cells were labeled with anti-BrdU (Becton Dickinson) and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G antibodies (Jackson Laboratories) according to Stewart et al. (31). DNA was stained with Hoechst 33258. Analysis was performed on a FACStar+ flow cytometer (Becton Dickinson).
Prostaglandin assays.
Prostaglandin E2
(PGE2) and 6-keto-prostglandin
F1
(6-keto-PGF1
)
concentrations in incubation media were determined, respectively, by
scintillation proximity and regular radioimmunoassays by using
commercially available kits (Amersham).
Cyclin D1 detection by Western blotting. BAC were seeded in 6-cm dishes at a density of 106 cells/plate and grown as described in Cell isolation and culture. After stimulation with the various effectors for the indicated periods of time, cells were lysed in 500-µl lysis buffer (20 mM tris(hydroxymethyl)aminomethane, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1 mM dithiothreitol, 100 µM Na3VO4, 50 nM okadaic acid, 25 µg/ml leupeptin, and 25 µg/ml aprotinin). Soluble proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide slab gels and transferred to a polyvinyl difluoride membrane. Blots were probed with a monoclonal anti-cyclin D1 antibody (Oncogene Science) which was revealed by enhanced chemiluminescence (Amersham). Blots were analyzed by laser densitometry.
Histone H1 kinase assay. Cell lysates obtained as described in Cyclin D1 detection by Western blotting were subjected to immunoprecipitation by addition of 2 µg of anti-cyclin D1 antibody during 1 h followed by incubation with 25-µl protein G sepharose beads. After extensive washing of the immunoprecipitates, histone H1 phosphorylation was assayed as described by Takase et al. (33). At the end of the reaction, soluble proteins were separated by SDS-PAGE and phosphorylated histone H1 was quantified by using a PhosphorImager and ImageQuant software (Molecular Dynamics).
Statistics. Data are reported as means ± SD of triplicate determinations. All experiments were performed at least three times in an independent fashion. Statistical analysis of the raw data was performed by analysis of variance followed by appropriate post hoc tests (Student's t-test, Scheffé's F-test). Unless otherwise indicated, values are taken as significant for values of P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BAC express both AT1 and AT2 receptors. Competition-binding experiments indicated that BAC express both AT1 and AT2 receptors. The AT2-selective agonist CGP-42112 competed for ~20% of the specific 125I-[Sar1,Ile8]ANG II binding sites with high affinity [inhibitory constant (Ki) = 0.3 ± 0.1 nM] and to the remaining sites with low affinity (Ki = 1.2 ± 0.3 µM), compatible with AT2 and AT1 receptors, respectively. The AT1-selective antagonist losartan competed for ~80% of the specific 125I-[Sar1,Ile8]ANG II binding sites with high affinity (Ki = 50 ± 12 nM) and to the remaining sites with low affinity (Ki = 5.7 ± 0.3 µM). ANG II also competed for 125I-CGP-42112 binding with high affinity (Ki = 0.4 ± 0.2 nM; data not shown). These data are in agreement with those reported by Ouali et al. (23) and are consistent with a previous study in which both receptor subtypes were found to be involved in the stimulation of cortisol production (10).
ANG II does not induce proliferation of quiescent BAC but inhibits bFGF-stimulated proliferation. To achieve quiescence, the cells were deprived of serum for 72 h. The number of cells did not vary significantly when cells were maintained in serum-free medium, indicating their quiescence. As shown in Fig. 1, exposure to ANG II resulted in a very weak but nonsignificant proliferative effect (P > 0.05). By contrast, bFGF induced a 2.6-fold increase of the cell population over the same period of time. This proliferation was inhibited by 52 ± 4.5% by concomitant but single-time addition of ANG II.
|
ANG II inhibits bFGF-stimulated [3H]thymidine incorporation. Figure 2 shows that under control conditions, [3H]thymidine incorporation is minimal and increases only slightly over time (maximum 60%), confirming that most cells are quiescent. Exposure of the cells to bFGF increased [3H]thymidine incorporation up to eightfold in a time-dependent fashion, with maximal incorporation being reached after 30-36 h.
|
ANG II blocks bFGF-stimulated cells in G1 phase. The effects of bFGF and ANG II treatments, alone or in combination, on the progression of the cells through the cycle were investigated by flow cytometry with simultaneous measurement of incorporated BrdU and total DNA. As shown in Fig. 3, after starvation, 90% of the cells were in G1, 5% in S, and 5% in G2 and M phases. After 30 h exposure to bFGF, the number of cells in the S phase increased fourfold, whereas the number of cells in G2 and M phases was unaffected. When the cells were exposed to bFGF in the presence of ANG II, the number of cells in the S phase dropped to the same level as that observed in cells treated with ANG II alone.
|
AT1- and AT2-selective analogs modulate bFGF-stimulated [3H]thymidine incorporation. To determine which ANG II-receptor subtype mediates inhibition of bFGF-stimulated DNA synthesis, we studied the effect of selective AT1 and AT2 analogs on [3H]thymidine incorporation. The results illustrated in Fig. 4 indicate that the inhibitory effect of ANG II is only partly blocked by the AT1 or AT2 antagonists losartan and PD-123319, respectively. Each of these analogs inhibits the effect of ANG II to a comparable extent (i.e., 43 and 38%, respectively) when added individually at the same concentration of 1 µM. The effect of losartan could be mimicked by another AT1-selective antagonist, valsartan (CGP-48933), at a 10-fold-lower concentration, in agreement with its higher affinity for the AT1 receptor (data not shown). The effect of ANG II was further partly mimicked by the AT2 agonist CGP-42112, which inhibited bFGF-stimulated [3H]thymidine incorporation by 47%, a level similar to that obtained with ANG II in the presence of the AT1 antagonists and to the results reported previously in endothelial and vascular smooth muscle (22, 32).
|
Pertussis toxin blocks AT1- and AT2-receptor-mediated inhibition of [3H]thymidine incorporation. Pretreatment of BAC with pertussis toxin for 48 h did not affect the ability of bFGF to stimulate [3H]thymidine incorporation but completely blocked the inhibition of this response either by ANG II alone or in the presence of the AT2 antagonist PD-123319, as well as by the AT2 agonist CGP-42112 (Fig. 5), indicating that both AT1- and AT2-receptor signaling pathways are triggered by Gi.
|
ANG II stimulates PGE2 production through AT1 receptors through a pertussis toxin-sensitive pathway. As shown in Table 1, ANG II was able to stimulate PGE2 production 5.3-fold when added alone and 5.5-fold when added in the presence of bFGF. This response was blocked by the AT1 antagonist losartan but was not affected by the AT2 antagonist PD-123319. The AT2 agonist CGP-42112 had no effect on PGE2 synthesis either.
|
Blockade of prostanglandin synthesis by indomethacin blunts AT1 receptor-mediated antiproliferation. Indomethacin, which decreased PGE2 synthesis to below control levels (Table 1), also completely abolished the AT1 receptor-mediated inhibition of bFGF-induced [3H]thymidine incorporation (ANG II in the presence of PD-123319) without, however, affecting AT2-mediated antiproliferation, as indicated by its lack of effect on the action of CGP-42112 and of ANG II in the presence of losartan (Fig. 6).
|
Exogenous prostaglandins inhibit bFGF-stimulated [3H]thymidine incorporation. As shown in Table 2, exogenous PGE2 and prostaglandin D2 (PGD2) both completely inhibited bFGF-stimulated [3H]thymidine incorporation. This antiproliferative effect was potentiated by ANG II in the presence of losartan, suggesting additive effects of prostaglandin- and AT2 receptor-mediated effects. This finding is in agreement with the putative involvement of cyclooxygenase stimulation and of increased PGE2 generation in AT1 receptor-mediated antiproliferation.
|
The PTP inhibitor sodium orthovanadate blocks AT2 receptor-mediated antiproliferation. Figure 7 shows that exposure of BAC to the PTP inhibitor sodium orthovanadate (50 µM) suppressed the growth-inhibitory effect of ANG II in the presence of losartan and of the AT2 agonist CGP-42112, whereas it did not alter the response to ANG II alone or in the presence of the AT2 antagonist PD-123319. Similar results were obtained with bpV(pic) (5 µM, data not shown), an organic derivative of vanadate that has been reported to be a potent and selective PTP inhibitor (28), suggesting that PTP(s) are involved in AT2- but not in AT1-receptor-mediated antiproliferation.
|
ANG II inhibits bFGF-stimulated cyclin D1 expression through AT1 and AT2 receptors. Expression of cyclin D1, which plays a major role in the progression of the cells through the G1 phase, was monitored by Western blotting. The monoclonal antibody used recognized a major protein band migrating with an apparent molecular mass of 36 kDa, corresponding to the reported mass of cyclin D1.
The blot shown in Fig. 8 indicates the ability of bFGF to strongly stimulate (>70-fold) cellular levels of cyclin D1 after 24-30 h of exposure. ANG II by itself enhanced expression of this protein (up to 14-fold) but significantly attenuated its induction by bFGF (53%). This attenuating effect is maintained in the presence of PD-123319, appearing slightly stronger at 30 h (82%). Selective AT2 receptor stimulation, achieved either with CGP-42112 or with ANG II in the presence of losartan, resulted in a more pronounced repression of cyclin D1 expression, with levels comparable to those found in ANG II-stimulated cells.
|
ANG II inhibits bFGF stimulated cyclin D1-dependent kinase activity through AT1 and AT2 receptors. Cyclin D1-dependent kinase activity, as assayed by measuring histone H1 phosphorylation by anti-cyclin D1 immunoprecipitates, was significantly increased (1.5-fold) in cells exposed to bFGF for 27 h (Fig. 9). This activity fell back to near control levels in the presence of ANG II. This inhibitory effect was mediated by both AT1- and AT2-receptor stimulation, as indicated by the ability of both PD-123319 and losartan to block it and of CGP-42112 to mimic it.
|
Indomethacin blunts AT1 receptor-mediated inhibition of cyclin D1-dependent kinase activity. Indomethacin, which inhibited PGE2 synthesis and abolished the AT1 receptor-mediated inhibition of bFGF-induced [3H]thymidine incorporation (Fig. 5), also suppressed the inhibitory effect of ANG II on bFGF stimulated cyclin D1-dependent kinase activity. Cyclooxygenase inhibition selectively affected the AT1 receptor-mediated action of ANG II, as indicated by the data obtained with the selective analogs CGP-42112 and PD-123319 (Fig. 9).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study shows that in BAC which have retained their
physiological steroidogenic responsiveness (10), ANG II inhibits growth
factor-stimulated proliferation. This observation is in contrast with the generally accepted growth-promoting action of the
vasoactive peptide in various tissues or cells (16), thus indicating
that ANG II can exert both positive and negative control of cell
proliferation. Dual regulation of cell growth by a single ligand in
different tissues is not unique to ANG II. The best known example is
that of transforming growth factor-1, which inhibits proliferation
of various epithelial cells but exerts mitogenic effects on certain
fibroblasts and tumor cells (12). Similarly, peptide hormones like
somatostatin (29) and endothelin (19) have also been shown to regulate
growth positively or negatively, and this has been attributed to their
interaction with different receptor subtypes linked to different
signaling pathways.
The growth-promoting actions of ANG II have been shown to be mediated by AT1 receptor-dependent signaling pathways (16), but two recent reports indicate that in cells also expressing AT2 receptors this peptide inhibits proliferation through an as yet undefined but AT2 receptor-linked pathway (22, 32). By analogy with the trophic factors mentioned above, it was tempting to speculate that the growth-regulatory effect of ANG II depends on the receptor subtype that is expressed, with the AT1 receptor mediating proliferation and the AT2 receptor being linked to inhibition of growth. As a result of this, modulation of the expression of AT1 and AT2 receptors would be the key parameter in determining the type of trophic response of a cell to the octapeptide. This hypothesis has been further supported by the recently reported ability of the AT2 receptor to switch off the proliferative response to ANG II when transfected in vascular smooth muscle cells both in vitro and in vivo (22).
A major finding of this work is that in adrenocortical cells, the antiproliferative effect of ANG II involves not only AT2 but also AT1 receptors. The antiproliferative effects mediated by both receptors are additive, suggesting that they occur through distinct transduction mechanisms, leading to an eventually identical cellular response.
Agonist binding to the AT1 receptor leads to coupling to the heterotrimeric G proteins Gi and Gq (8). In the adrenal fasciculata, like in most other tissues, the AT1 signaling pathway, which mediates one of the major functions of these cells, i.e., steroidogenesis, involves activation of the Gq-phospholipase C (PLC)-protein kinase C (PKC) cascade (9, 24). Desensitization of this pathway by downregulation of PKC with the active phorbol ester phorbol 12-myristate 13-acetate did not, however, affect the antiproliferative action of ANG II (data not shown), thus excluding the involvement of this cascade in this response. The AT1 receptor has also been reported to interact with Gi, resulting in the inhibition of adenylate cyclase (2, 8, 24). We show here that pretreatment of BAC with pertussis toxin, known to inactivate Gi, but not Gq, through ADP ribosylation, completely abolished the AT1-mediated antiproliferative action. Pertussis toxin has also been reported to inhibit ANG II-stimulated PGE2 synthesis in mesangial cells, which express only AT1 receptors (27), suggesting the involvement of a member of the Gi family in this signaling pathway. Because prostaglandins have previously been reported to inhibit the proliferation of BAC (11), we examined the effect of the cyclooxygenase inhibitor indomethacin on the growth-inhibitory action of ANG II in BAC. We found that this compound selectively blunted AT1- and not AT2-mediated antiproliferation. In agreement with this observation, we found that ANG II stimulates PGE2 production by these cells. This effect is also purely AT1 dependent and is inhibited by pertussis toxin.
To verify the hypothesis that PGE2 is indeed involved in AT1-mediated antiproliferation, we tested the effect of exogenous prostaglandins on bFGF-stimulated growth. We found that PGE2, as well as PGD2, inhibited bFGF-stimulated DNA synthesis to control levels. The additive effects of exogeneous prostaglandins and selective AT2 receptor stimulation indicate that AT1- and AT2-receptor-mediated effects involve distinct pathways, which contribute in an independent fashion to inhibition of growth.
Taken together, these data suggest that, in BAC, AT1 receptors stimulate prostaglandin synthesis, in particular PGE2, and that this response leads to inhibition of growth factor-stimulated proliferation. This hypothesis is consistent with other studies reporting antiproliferative effects of PGE2 in vascular (35) and tracheal (13) smooth muscle cells as well as in lymphocytes (30).
The mechanism through which the
AT1 receptor stimulates
PGE2 synthesis in these cells
remains to be clarified, but inhibition of this pathway by pertussis
toxin suggests the involvement of Gi activating phospholipase
A2, most probably through its
-subunits, rather than of the
Gq-PLC-PKC pathway.
PGE2 itself is thought to mediate
its effects through stimulation of adenylate cyclase, a mechanism that
has been shown to induce inhibition of growth factor activation of
mitogen-activated protein (MAP) kinase (36) and that has also been
proposed to be responsible for the antiproliferative effects of ACTH in
adrenal cells (7).
As mentioned earlier, two recent reports indicate that the AT2 receptor mediates inhibition of proliferation (22, 32), but the pathway leading to this response has not been clarified.
The current knowledge of the signaling mechanisms linked to this receptor is still very preliminary, but it appears that they are clearly distinct from those used by the AT1 receptor (2, 8). We reported earlier that this receptor decreases cellular guanosine 3',5'-cyclic monophosphate through inhibition of ANP receptor guanylate cyclase activity (4) and modulates T-type Ca2+ currents (6). Ligand binding to this receptor has also been shown to stimulate PTP activity in different cell lines (5, 21), and this mechanism appears to be required for AT2 inhibition of guanylate cyclase (4) and T-type calcium currents (6), indicating that it is a proximal event in the transduction pathways linked to this receptor.
Considering the major role of protein tyrosine phosphorylation in the mitogenic cascades, it was tempting to speculate that PTP activation could be involved in the AT2 receptor-mediated antiproliferative effects of ANG II. In agreement with this hypothesis, we found the PTP inhibitor sodium orthovanadate and its more potent organic analog bpV(pic) (28) to completely block AT2-mediated inhibition of bFGF-stimulated DNA synthesis without affecting the AT1-dependent response. Interestingly, sodium orthovanadate has also been reported to attenuate AT2 receptor-mediated induction of apoptosis (37), possibly by inhibiting MAP kinase phosphatase 1 (MKP-1). Apoptosis is, however, unlikely to contribute to the antiproliferative effect in BAC because we were unable to detect any significant increase of apoptotic cell number after exposure to ANG II (data not shown).
Two other peptides, somatostatin and dopamine, have also been reported to mediate antiproliferative actions through pathways involving PTP activation (14, 25). Both appear to activate PTPs through a Gi-dependent mechanism. In contrast to this, AT2 receptor activation of PTP has been reported to be G protein independent, and from a global point of view the interaction of this receptor with G proteins is still a matter of debate. We, therefore, verified the effect of pertussis toxin on AT2-mediated antiproliferation and found this treatment to block this response as effectively as the AT1 response. This suggests that in these cells AT2 receptor-dependent inhibition of growth requires the functional integrity of Gi.
Thus, in BAC, both ANG II receptor subtypes trigger distinct signaling cascades, which ultimately, however, contribute to a common cellular response: antiproliferation.
To determine the level at which both transduction pathways converge, we first investigated the effect of ANG II on progression through the cell cycle. Fluorescence-activated cell sorter (FACS) analysis indicated that the octapeptide arrests bFGF-stimulated cells in G1 phase. This suggests that interference with the growth factor-stimulated mitogenic cascade could occur at the level of MAP kinase activation, which is believed to be required only for entry into G1, amongst others, by inducing the expression of cyclin D (18). Cyclins act as regulatory subunits of kinases called cyclin-dependent kinases (CDKs), which are required for progression of the cells through the mitotic cycle. Whereas the different CDKs are expressed throughout the cell cycle, the various cyclins, which are required for their activation, are expressed only at specific stages and are degraded rapidly thereafter. Cyclins D and E are specific of the G1 phase, with cyclin D accumulating in mid-G1 before the appearance of cyclin E, which is believed to be required for the transition of the G1 to S phase.
We measured the kinase activity of the cyclin D1-associated CDKs after stimulation of the cells with bFGF and found it to be blunted by ANG II. This inhibitory effect appears to be mediated by both AT1 and AT2 transduction pathways, as indicated by experiments using the subtype-selective ligands. The ability of indomethacin to selectively block the AT1-dependent inhibition confirms the importance of prostaglandin synthesis in AT1-mediated antiproliferation in these cells.
To analyze the intermediate steps leading to the observed decrease in CDK activity, we measured the expression of cyclin D1. Consistent with the data on CDK activity, we found ANG II to significantly inhibit bFGF-induced cyclin D1 expression. A similar level of inhibition was achieved by selective AT1 receptor activation. Agonist binding to the AT2 receptor, however, suppressed cyclin D1 expression to near control levels. As mentioned earlier, CDK activation requires its interaction with specific cyclins, which in turn is dependent on their expression. Regulation of CDK activity appears to be much more complex than initially thought. Recent data indicate that it not only depends on binding of the appropriate cyclin but is also regulated by other proteins that can inhibit the catalytic activity of the cyclin-CDK complexes through direct interaction. It is thus conceivable that AT1 and AT2 signaling cascades modulate CDK activity through different mechanisms.
Whereas the AT2 receptor appears to block CDK activity essentially through repression of cyclin D1 expression, the AT1 receptor might mediate its action through the induction of an inhibitory protein. This would be consistent with the previously reported ability of adenosine 3',5'-cyclic monophosphate to raise cellular levels of p27, which binds to and inhibits G1 cyclin-CDK complexes (17).
In contrast to the present study, ANG II has previously been reported
to exert proliferative actions in adrenal cells (9, 15), although this
response was variable between the selected clones of cells (15). It is
worthwhile noting that the culture and starving conditions applied in
other studies were different and in particular that the duration of
culture before experimentation was longer. It is well known that
culture conditions and time affect a variety of cellular responses, and
Tian et al. (34) reported earlier that the mitogenic action of ANG II
on adrenal cells appeared only after at least 5 days of culture,
suggesting that the cells undergo changes in their state of
differentiation. Of particular interest with regard to these changes is
the progressive decrease in Gi
in cultured BAC, resulting in the loss of ability of ANG II to inhibit
adenylate cyclase in these cells (1). Moreover, the authors found that,
in parallel, the other signaling pathway the
AT1 receptor is coupled to, i.e.,
the PLC-PKC cascade, becomes functionally dominant, thus accounting for
the ability of ANG II to potentiate instead of inhibit adenylate
cyclase.
This observation is consistent with a potential loss of both AT1- and AT2-receptor-mediated antiproliferative pathways that we found to occur through Gi and that could account for the unmasking of a proliferative effect due to activation of the Gq-PLC-PKC pathway. This latter pathway is maintained even after prolonged culture (1) and is responsible for ANG II-stimulated activation of MAP kinase (7). These observations stress the importance of assessing the differentiation state of the cells and the integrity of the signal transduction mechanisms before investigating the often complex growth-regulatory mechanisms.
This study points to the complexity of the regulatory mechanisms that determine the trophic actions of ANG II. The growth-regulatory actions of this peptide appear to depend not only on the receptor subtypes which are expressed, but also, and essentially, on the intracellular environment that determines the signaling pathways they are coupled to. Our data indicate that in adrenal cells the AT2 receptor triggers a PTP-activating cascade that inhibits cyclin D1 expression. The AT1 receptor mediates stimulation of prostaglandin synthesis, resulting in the inhibition of cyclin D1-associated CDK activity. Both pathways exert additive effects leading to arrest of the cells in G1 phase. Interestingly, the AT1 receptor, as in other cell types, is also linked to a MAP kinase-activating cascade, which can lead to proliferation when the inhibitory pathways are disabled. It appears thus that the type and the differentiation state of the cell determine the balance between the growth-promoting and -inhibitory cascades linked to ANG II receptors and eventually define its ultimate biological response, proliferation or quiescence.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to C. Blanc-Brude and I. Gaillard for the preparation of primary cultures of bovine adrenocortical cells and to G. Oddoz for the prostaglandin assays. We thank N. Bertacchi and D. Grunwald for their help during the FACS measurements. We thank Dr. M. de Gasparo for providing us with CGP-42112 and valsartan (CGP-48933) (Ciba-Geigy). Losartan (DuP-753) was a generous gift from DuPont and PD-123319 from Parke-Davis. Potassium bisperoxo(pyridine-2-carboxylato)oxovanadate was a generous gift of Dr. B. I. Posner and Dr. A. Shaver (McGill University, Montreal, PQ, Canada).
![]() |
FOOTNOTES |
---|
This work was supported by Institut National de la Santé et de la Recherche Médicale, the Commissariat à l'Energie Atomique, the Association pour la Recherche sur le Cancer, and the Ligue Nationale contre le Cancer.
Address for reprint requests: S. P. Bottari, Laboratoire de Biochimie A, Centre Hospitalier Universitaire de Grenoble, B.P. 217, 38043 Grenoble Cedex 9, France.
Received 4 December 1996; accepted in final form 12 May 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Begeot, M.,
D. Langlois,
A. Penhoat,
and
J. M. Saez.
Variations in guanine-binding proteins (Gs, Gi) in cultured bovine adrenal cells.
Eur. J. Biochem.
174:
317-321,
1988[Abstract].
2.
Bottari, S. P.,
M. de Gasparo,
U. M. Steckelings,
and
N. R. Levens.
Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implication.
Front. Neuroendocrinol.
14:
123-171,
1993[Medline].
3.
Bottari, S. P.,
V. Taylor,
I. N. King,
Y. Bogdal,
S. Whitebread,
and
M. de Gasparo.
Angiotensin II AT2 receptors do not interact with guanine nucleotide binding proteins.
Eur. J. Pharmacol. Mol. Pharmacol. Sect.
207:
157-163,
1991[Medline].
4.
Brechler, V.,
N. R. Levens,
M. de Gasparo,
and
S. P. Bottari.
Angiotensin AT2 receptor mediated inhibition of particulate guanylate cyclase: a link with tyrosine phosphatase stimulation?
Recept. Channels
2:
89-97,
1994[Medline].
5.
Brechler, V.,
S. Reichlin,
M. de Gasparo,
and
S. P. Bottari.
Angiotensin II stimulates tyrosine phosphatase activity through a G-protein independent mechanism.
Recept. Channels
2:
79-87,
1994[Medline].
6.
Buisson, B.,
L. Laflamme,
S. P. Bottari,
M. de Gasparo,
N. Gallo-Payet,
and
M. D. Payet.
A G protein is involved in the angiotensin AT2 receptor inhibition of the T-type calcium current in non-differentiated NG108-15 cells.
J. Biol. Chem.
270:
1670-1674,
1995
7.
Chabre, O.,
F. Cornillon,
S. P. Bottari,
E. M. Chambaz,
and
I. Vilgrain.
Hormonal regulation of mitogen-activated protein kinase activity in bovine adrenocortical cells: cross-talk between phosphoinositides, adenosine 3',5'-monophosphate and tyrosine kinase receptor pathways.
Endocrinology
136:
956-964,
1995[Abstract].
8.
Clauser, E. L.,
K. M. Curnow,
S. Conchon,
E. Davies,
B. Teutsch,
B. Vianello,
C. Monnot,
and
P. Corvol.
Molecular structure and mechanisms of action of mammalian angiotensin II receptors.
Curr. Opin. Endocrinol. Diabetes
2:
404-411,
1995.
9.
Clyne, C. D.,
M. R. Nicol,
S. MacDonald,
B. C. Williams,
and
S. W. Walker.
Angiotensin II stimulates growth and steroidogenesis in zona fasciculata/reticularis cells from bovine adrenal cortex via the AT1 receptor subtype.
Endocrinology
132:
2206-2212,
1993[Abstract].
10.
Defaye, G.,
S. Lecomte,
E. M. Chambaz,
and
S. P. Bottari.
Stimulation of cortisol production through angiotensin AT2 receptors in bovine fasciculata cells.
Endocr. Res.
21:
183-187,
1995[Medline].
11.
Duperray, A.,
and
E. M. Chambaz.
Effect of prostaglandin E1 and ACTH on proliferation and steroidogenic activity of bovine adreno-cortical cells in primary culture.
J. Steroid Biochem.
13:
1359-1364,
1980[Medline].
12.
Fausto, N.
Multifunctional roles for transforming growth factor-1.
Lab. Invest.
65:
497-498,
1991[Medline].
13.
Florio, C.,
J. G. Martin,
A. Styhler,
and
S. Heisler.
Antiproliferative effect of prostaglandin E2 in cultural guinea pig tracheal smooth muscle cells.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L131-L137,
1994
14.
Florio, T.,
M. G. Pan,
B. Newman,
R. E. Hershberger,
O. Civelli,
and
P. J. Stork.
Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity.
J. Biol. Chem.
267:
24169-24172,
1992
15.
Gill, G. N.,
C. R. Ill,
and
M. H. Simonian.
Angiotensin stimulation of bovine adrenocortical cell growth.
Proc. Natl. Acad. Sci. USA
74:
5569-5573,
1977[Abstract].
16.
Huckle, W. R.,
and
H. S. Earp.
Regulation of cell proliferation and growth by angiotensin II.
Prog. Growth Factor Res.
5:
177-194,
1994[Medline].
17.
Kato, J.,
M. Matsuoka,
K. Polyak,
J. Massagué,
and
C. J. Sherr.
Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27kip1) of cyclin-dependent kinase 4 activation.
Cell
79:
487-496,
1994[Medline].
18.
Lavoie, J. N.,
G. L'Allemain,
A. Brunet,
R. Müller,
and
J. Pouysségur.
Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway.
J. Biol. Chem.
271:
20608-20616,
1996
19.
Mallat, A.,
L. Fouassier,
A. M. Préaux,
C. Serradeil-Le Gal,
D. Raufaste,
J. Rosenbaum,
D. Dhumeaux,
C. Jouneaux,
P. Mavier,
and
S. Lotersztajn.
Growth inhibitory properties of endothelin-1 in human hepatic myofibroblastic Ito cells.
J. Clin. Invest.
96:
42-49,
1995[Medline].
20.
Meffert, S.,
M. Stoll,
U. M. Steckelings,
S. P. Bottari,
and
T. Unger.
The angiotensin AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells.
Mol. Cell. Endocrinol.
122:
59-67,
1996[Medline].
21.
Nahmias, C.,
S. Cazaubon,
M. Briend-Sutren,
D. Lazard,
P. Villageois,
and
A. D. Strosberg.
Angiotensin II AT2 receptors are functionally coupled to protein tyrosine dephosphorylation in NIE-115 neuroblastoma cells.
Biochem. J.
306:
87-92,
1995[Medline].
22.
Nakajima, M.,
H. G. Hutchinson,
M. Fujinaga,
W. Hayashida,
R. Morishita,
L. Zhang,
M. Horiuchi,
R. E. Pratt,
and
V. J. Dzau.
The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.
Proc. Natl. Acad. Sci. USA
92:
10663-10667,
1995[Abstract].
23.
Ouali, R.,
M. C. LeBrethon,
and
J. M. Saez.
Identification and characterization of angiotensin II receptor subtypes in cultured bovine and human adrenal fasciculata cells and PC12W cells.
Endocrinology
133:
2766-2772,
1993[Abstract].
24.
Ouali, R.,
S. Poulette,
A. Penhoat,
and
J. M. Saez.
Characterization and coupling of angiotensin II receptor subtypes in cultured bovine adrenal fasciculata cells.
J. Steroid Biochem. Mol. Biol.
43:
271-280,
1992[Medline].
25.
Pan, M. G.,
T. Florio,
and
P. J. Stork.
G protein activation of a hormone-stimulated phosphatase in human tumor cells.
Science
256:
1215-1217,
1992[Medline].
26.
Penhoat, A.,
C. Jaillard,
A. Crozat,
and
J. M. Saez.
Regulation of angiotensin II receptors and steroidogenic responsiveness in cultured bovine fasciculata and glomerulosa adrenal cells.
Eur. J. Biochem.
172:
247-254,
1988[Abstract].
27.
Pfeilschifter, J.,
and
C. Bauer.
Pertussis toxin abolishes angiotensin II-induced phosphoinositide hydrolysis and prostaglandin synthesis in rat mesangial cells.
Biochem. J.
236:
289-294,
1986[Medline].
28.
Posner, B. I.,
R. Faure,
J. W. Burgess,
A. P. Bevan,
L. Lachance,
G. Zhang-Sun,
I. G. Fantus,
J. B. Ng,
D. A. Hall,
B. Soo-Lum,
and
A. Shaver.
Peroxovanadium compounds.
J. Biol. Chem.
229:
4596-4604,
1994.
29.
Ruiz-Torres, P.,
F. J. Lucio,
M. Gonzalez-Rubio,
M. Rodriguez-Puyol,
and
D. Rodriguez-Puyol.
A dual effect of somatostatin on the proliferation of cultured rat mesangial cells.
Biochem. Biophys. Res. Commun.
195:
1057-1062,
1993[Medline].
30.
Santos-Neto, L.,
C. E. Tosta,
and
J. G. Dorea.
Zinc reverses the increased sensitivity of lymphocytes from aged subjects to the antiproliferative effect of prostaglandin E2.
Clin. Immunol. Immunopathol.
64:
184-187,
1992[Medline].
31.
Stewart, N.,
G. G. Hicks,
F. Paraskevas,
and
M. Mowat.
Evidence for a second cell cycle block at G2/M by p53.
Oncogene
10:
109-115,
1995[Medline].
32.
Stoll, M.,
U. M. Steckelings,
M. Paul,
S. P. Bottari,
R. Metzger,
and
T. Unger.
The angiotensin AT2-receptors mediates inhibition of cell proliferation in coronary endothelial cell.
J. Clin. Invest.
95:
651-657,
1995[Medline].
33.
Takase, K.,
N. Terada,
A. Szepsi,
H. Treraoka,
E. W. Gelfand,
and
J. J. Lucas.
Release from G0/G1 arrest induced by dimethylsulfoxide in human lymphoid cells: regulation of synthesis and activation of the p33cdk2 and p34cdc2 kinases.
Cell Growth Differ.
5:
1051-1059,
1994[Abstract].
34.
Tian, Y.,
R. E. Balla,
A. J. Baukal,
and
K. J. Catt.
Growth responses to angiotensin II in bovine adrenal glomerulosa cells.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E135-E144,
1995
35.
Uehara, Y.,
T. Ishimitsu,
H. Kimura,
M. Ishii,
and
T. Sugimoto.
Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats.
Prostaglandins
36:
847-857,
1988[Medline].
36.
Wu, J.,
P. Dent,
T. Jelinek,
A. Wolfman,
M. J. Weber,
and
T. W. Sturgill.
Inhibition of EGF-activated MAP kinase signalling pathway by adenosine 3',5'-monophosphate.
Science
262:
1065-1069,
1993[Medline].
37.
Yamada, T.,
M. Horiuchi,
and
V. J. Dzau.
Angiotensin II type 2 receptor mediates programmed cell death.
Proc. Natl. Acad. Sci. USA
93:
156-160,
1996