ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells

Panagiotis Liakos1, Nicolas Bourmeyster1,2, Geneviève Defaye1, Edmond M. Chambaz1,2, and Serge P. Bottari1,2

1 Institut National de la Santé et de la Recherche Médicale, Unité 244, Centre d'Etudes Nucléaires de Grenoble and 2 Laboratoire de Biochimie A, Centre Hospitalier Universitaire de Grenoble, 38043 Grenoble Cedex 9, France

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

Proliferation was determined in triplicate 22-mm wells. BAC were seeded at a density of 105 cells/well. Quiescent cells were incubated in fresh Ham's F-12 containing 0.1% BSA with the effectors during 96 h. Cells were recovered with trypsin-EDTA and counted with a Coulter counter. For both types of experiments, effectors were added only once.

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 F1alpha (6-keto-PGF1alpha ) 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
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Abstract
Introduction
Methods
Results
Discussion
References

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.


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Fig. 1.   Effect of angiotensin II (ANG II) on basic fibroblast growth factor (bFGF)-induced proliferation. After starvation for 72 h, cells were incubated during 96 h with medium alone or medium containing bFGF (10 ng/ml), ANG II (3 nM), or bFGF and ANG II together. After trypsinolysis, cells were counted. Data are means ± SD of the normalized data obtained in 3 independent experiments. All measurements were performed in triplicate in each experiment. t0, time = 0 h; t 96h, time = 96 h. Statistical analysis indicates that bFGF is significantly different from all other conditions (P < 0.05).

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.


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Fig. 2.   Effect of ANG II on bFGF-induced [3H]thymidine incorporation. Quiescent cells were incubated for 24, 27, 30, 36, and 48 h in Ham's F-12 medium with 0.1% bovine serum albumin (BSA) alone (open bars) or in presence of ANG II (3 nM; hatched bars), bFGF (10 ng/ml; solid bars), or ANG II and bFGF together (crosshatched bars). [3H]thymidine incorporation was allowed for the last 3 h of incubation and assayed as described in METHODS. Each value is mean ± SD of triplicate measurements in same experiment. Data shown are representative of 3 independent experiments. cpm, Counts/min.

ANG II by itself only stimulated [3H]thymidine incorporation up to 56% above control levels but inhibited bFGF-induced incorporation by 90% (P < 0.01) between 30 and 36 h, indicating that the octapeptide inhibits growth factor-induced DNA synthesis. This inhibitory effect persisted up to at least 42 h after addition of the effectors, suggesting arrest of the cells in G1 phase or during G1/S transition.

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.


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Fig. 3.   Effect of bFGF, ANG II, and bFGF plus ANG II on progression of bovine adrenal fasciculata cells (BAC) through the cell cycle. Quiescent cells were incubated for 30 h in Ham's F-12 medium with 0.1% BSA alone (control; A) or in the presence of bFGF (10 ng/ml; B), ANG II (3 nM; C), or ANG II and bFGF (D), and 5-bromo-2'-deoxyuridine (BrdU) was allowed to incorporate during last 20 min of incubation. Subsequently, cells were stained with anti-BrdU fluorescein isothiocyanate-conjugated antibody. Total DNA was stained with Hoechst 33258. Cells were analyzed by using a FACStar+ cytometer. a: Distribution of BrdU incorporation in BAC as function of total DNA fluorescence. b: Distribution of cells as function of total DNA. Data shown are representative of 4 independent experiments.

The percentage of cells in G2 and M phases was not affected by either treatment. These observations confirm the data obtained with [3H]thymidine indicating that the cells are arrested before their entry in the S phase. The level of inhibition of progression into the S phase is also compatible with the degree of inhibition of proliferation illustrated in Fig. 1.

On the other hand, we were unable to detect a significant proportion of apoptotic cells under any of the various conditions tested by using this technique.

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).


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Fig. 4.   Effect of ANG II and its selective analogs on bFGF-induced [3H]thymidine incorporation. Quiescent cells were incubated for 30 h in Ham's F-12 medium with 0.1% BSA alone (control) or in presence of indicated compounds: ANG II (3 nM) bFGF (10 ng/ml), CGP-42112 (CGP; 1 µM), PD-123177 (PD; 1 µM), losartan (Los; 1 µM). [3H]thymidine was allowed to incorporate during the last 3 h. Data shown are means ± SD of normalized data obtained in 5 independent experiments. All measurements were performed in triplicate in each experiment. Statistical analysis indicates that bFGF and bFGF + ANG II + Los + PD are significantly different from all other conditions (P < 0.05) and that bFGF + ANG II + PD, bFGF + CGP, and bFGF + ANG II + Los are significantly different from control, ANG II, and bFGF + ANG II (P < 0.05).

The inhibitory effect of ANG II on bFGF-induced DNA synthesis was completely abolished by simultaneous addition of losartan and PD-123319.

These findings indicate that both AT1 and AT2 receptors contribute in an additive fashion to the antimitogenic action of ANG II.

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.


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Fig. 5.   Effect of pertussis toxin on [3H]thymidine incorporation. Cells were deprived during 72 h, and pertussis toxin (100 ng/ml) and NAD (10 µM) were added during the last 48 h. Cells were then incubated during 30 h with indicated compounds: bFGF (10 ng/ml), ANG II (3 nM), CGP (1 µM), PD (1 µM), Los (1 µM). [3H]thymidine was added during the last 3 h. Control values in the presence of pertussis toxin were 59 ± 11% of values obtained under standard conditions. Data shown are means ± SD of normalized data obtained in 3 independent experiments. All measurements were performed in triplicate in each experiment. Statistical analysis indicates that ANG II is significantly different from control and from all other conditions (P < 0.01). Other conditions did not vary significantly from each other (P > 0.05).

Pertussis toxin treatment also resulted in the appearance of a proliferative response to ANG II, as evidenced by a threefold (P < 0.02) increase in [3H]thymidine incorporation, as opposed to control conditions where the octapeptide by itself was ineffective in stimulating growth, suggesting that ANG II also has mitogenic properties that are repressed by Gi-dependent mechanisms.

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.

                              
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Table 1.   Regulation of prostaglandin E2 production by bFGF, ANG II, and its selective agonists or antagonists: effect of indomethacin, pertussis toxin, and orthovanadate

Treatment of the cells with pertussis toxin did not affect basal or bFGF-induced prostaglandin synthesis but resulted in a significant inhibition of AT1-receptor-mediated stimulation (68%). In contrast, the PTP inhibitor sodium orthovanadate significantly increased basal (2.2-fold) and stimulated (1.3- to 2.5-fold) PGE2 synthesis (Table 1).

The production of prostaglandin I2, determined by measuring the concentration of its major metabolite 6-keto-PGF1alpha , did not vary significantly in response to ANG II (data not shown).

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).


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Fig. 6.   Effect of indomethacin on modulation by ANG II and its selective analogs of bFGF-induced [3H]thymidine incorporation. Quiescent cells were incubated for 30 h in Ham's F-12 medium with 0.1% BSA and indomethacin (10 µM) alone (control) or in presence of indicated compounds: ANG II (3 nM) bFGF (10 ng/ml), CGP (1 µM), PD (1 µM), Los (1 µM). [3H]thymidine was allowed to incorporate during last 3 h. Indomethacin was added 1 h before incubation with the various compounds. Control values in the presence of indomethacin were 95 ± 17% of the values obtained under standard conditions. Data shown are means ± SD of normalized data obtained in 3 independent experiments. All measurements were performed in triplicate in each experiment. Statistical analysis indicates that bFGF and bFGF + ANG II + PD are significantly different from all other conditions (P < 0.01), which do not vary significantly (P > 0.05). bFGF is not significantly different from bFGF + ANG II + PD (P > 0.05).

These findings suggest that the AT1 receptor-dependent antiproliferative action of ANG II requires cyclooxygenase activity and, therefore, most probably is mediated by prostaglandins.

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.

                              
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Table 2.   Effect of prostaglandins E2 and D2 on bFGF-induced [3H]thymidine incorporation

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.


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Fig. 7.   Effect of orthovanadate on modulation by ANG II and its selective analogs of bFGF-induced [3H]thymidine incorporation. Quiescent cells were incubated for 30 h in Ham's F-12 medium with 0.1% BSA and sodium orthovanadate (50 µM) alone (control) or in presence of indicated compounds: ANG II (3 nM) bFGF (10 ng/ml), CGP (1 µM), PD (1 µM), Los (1 µM). [3H]thymidine was allowed to incorporate during the last 3 h. Orthovanadate was added 1 h before incubation with various compounds. Control values in the presence of orthovanadate were 96 ± 20% of values obtained under standard conditions. Data shown are means ± SD of normalized data obtained in 3 independent experiments. All measurements were performed in triplicate in each experiment. Statistical analysis indicates that bFGF, bFGF + CGP, and bFGF + ANG II + Los are significantly different from all other conditions (P < 0.05), which do not vary significantly (P > 0.05) from each other. bFGF, bFGF + CGP, and bFGF + ANG II + Los are not significantly different from each other (P > 0.05).

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.


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Fig. 8.   Western blot analysis of the regulation of cyclin D1 expression by bFGF, ANG II, and its selective analogs. Quiescent cells were incubated for 24, 27, or 30 h in Ham's F-12 medium with 0.1% BSA alone (control) or in the presence of indicated compounds: ANG II (3 nM) bFGF (10 ng/ml), CGP (1 µM), PD (1 µM), Los (1 µM). At end of indicated incubation times, cells were lysed and soluble proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted. A: cyclin D1 was detected by using a monoclonal antibody and enhanced chemiluminescence as described in METHODS. B: laser densitometric analysis of Western blots performed as described above. Data shown are means of values obtained with 2 blots.

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.


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Fig. 9.   Effect of indomethacin on regulation by ANG II and its selective analogs of bFGF-stimulated cyclin D1-associated cyclin-dependent kinase activity. Quiescent cells were incubated for 27 h in Ham's F-12 medium with 0.1% BSA alone (control) or in the presence of indicated compounds: ANG II (3 nM), bFGF (10 ng/ml), CGP (1 µM), PD (1 µM), Los (1 µM). At the end of indicated incubation times, cells were lysed and soluble proteins were subjected to immunoprecipitation with an anti-cyclin D1 monoclonal antibody. Kinase activity of immunoprecipitates was assayed using histone H1 as a substrate as described in METHODS. Hatched bars, control conditions; solid bars, in the presence of indomethacin (10 µM). Results are expressed in arbitrary units obtained with a PhosphorImager.

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-beta 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 beta gamma -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 Gialpha 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(4):C1324-C1334
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