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Regulated expression of angiotensin II (AT2) binding sites in R3T3 cells
Regulatory Peptides, 44 (1993) 199-206 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
199
REGPEP 01278
Regulated expression of angiotensin II (AT2) binding sites in R3T3 cells D a v i d T. D u d l e y and Rochelle M. Summerfelt Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI (USA) (Received 8 October 1992; accepted 11 December 1992)
Key words: PD 123319; Receptor; Growth factor; Mitogen; Fibroblast Summary R3T3 cells are a fibroblast cell line found to selectively express the AT 2 subtype of angiotensin II binding sites. We have previously shown that in these cells, the AT 2 sites do not appear to be coupled to G-proteins, do not modulate any of the common second messenger pathways associated with activation of angiotensin II receptors, and do not internalize bound ligand. Here we report that expression of AT 2 sites in these cells are subject to modulation by a variety of conditions. In actively growing cells the expression of AT 2 sites is very low, while in confluent, quiescent cells, the expression of AT 2 sites is markedly increased. Addition of serum, or growth factors, to quiescent cells causes a rapid decrease in the number of cell-surface AT 2 sites. Further, incubation of cells with ligands that bind to AT 2 sites causes a marked increase in the number of these sites in a time and dose dependent manner indicating homologous up-regulation of expression. These results indicate that expression of AT 2 sites in R3T3 cells is under sensitive and rapid control and further indicate that these cells may be an excellent model for studying the physiological regulation of expression of these sites.
Introduction Correspondence to: D.T. Dudley, Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, WarnerLambert Company, Ann Arbor, MI 48105, USA Abbreviations: bFGF, basic fibroblast growth factor; DMEM, Dulbeeco's Modified Essential Medium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; BSA, bovine serum alburnin; MEM, Minimal Essential Medium.
Angiotensin II is an eight amino acid peptide hormone known to have a variety of functions related to control of blood pressure and fluid homeostasis [ 1]. As a circulating hormone, many of its actions are thought to be mediated through cell-surface receptors. With the recent development of non-peptide
200
ligands that compete for binding with radiolabeled angiotensin II, it has become apparent that at least two major classes of binding sites or receptors exist for this peptide hormone. The AT 1 class, recognized by DuP 753 (also known as losartan), is commonly known as the angiotensin II receptor and cloning experiments have revealed it to be a member of the rhodopsin family of G-protein coupled receptors [2,3]. This receptor is typically coupled through phospholipase C and is responsible for essentially all of the currently known physiological actions of angiotensin II [4,5]. The AT 2 class is recognized by PD 123319 or CGP 42112A [4,6] and at present is not solely responsible for any known physiological actions of angiotensin II. Moreover, studies in cultured cells selectively expressing these sites have indicated that this site is not coupled through G-proteins and does not stimulate pathways typically associated with AT 1 receptors such as phosphatidylinositol turnover and inhibition of adenylate cyclase [7,8]. However, experiments in cultured neurons have revealed angiotensin II mediated decreases in cyclic G M P that are accounted for through AT 2 sites [9]. Recently, this effect has been attributed to changes in tyrosine phosphorylation, possibly through phosphatase activity [ 10]. While provocative, this has not been confirmed by other workers [11]. In adult animals, the AT 2 site is typically found in adrenal medulla [ 12], female reproductive organs [4] and certain brain regions [ 13]; areas that are largely distinct from where the AT 1 receptor is found. One of the most intriguing aspects of this site is its extensive localization in fetal tissue [ 14], and its rapid disappearance shortly after birth. We previously reported the finding of a line of fibroblast cells that selectively expressed AT 2 binding sites [7]. While these cells do not exhibit any detected perturbation in common signaling pathways upon exposure to angiotensin II, we report here that the expression of cell-surface AT 2 binding sites is dependent upon the growth state of the cells, and appears to be under rapid and sensitive control of
both mitogenic agents and ligands that bind to the site.
Materials and Methods
Materials [125I]Angiotensin II (2200 Ci/mmot) and [125I]Sarl,IleS-angiotensin II (2200 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Peptides were obtained from either Bachem Fine Chemicals (Torrance, CA), or Peninsula Laboratories (Belmont, CA). PD 123319, PD 123177 and DuP 753 were prepared as described [4]. Basic fibroblast growth factor (bFGF) was obtained from Intergen (Purchase, NY). Cell culture reagents were obtained from Gibco (Grand Island, NY) and fetal bovine serum from HyClone Laboratories (Logan, UT). Cell culture R3T3 cells, a mouse fibroblast line, were cultured as described [7] using Dulbecco's modified essential medium supplemented with 10~o fetal bovine serum. All experiments used cells grown as a monolayer. Cells were enumerated using a Coulter counter after release from plastic substrates with trypsin. Angiotensin H binding assay For most experiments, R3T3 cells were grown in 12-well plates and treated as indicated. Following treatment, cells were washed twice with 0.5 ml phosphate-buffered saline and incubated at 4°C for 3 rain in acid-glycine buffer (150 mM NaC1, 50 mM glycine, 1~o BSA, pH 3) in order to remove any ligands bound to cell-surface AT 2 binding sites. Cells were then rewashed twice with phosphate-buffered saline and incubated with minimal essential medium (MEM) supplemented with 20 mM Hepes, 0.1 ~o BSA, 0.2~o bacitracin and [125I]angiotensin II for 2 h at 37°C. Cells were then washed twice with icecold phosphate-buffered saline, solubilized in 2~o sodium dodecyl sulfate and the solubilized material
201
counted for 7 radioactivity. Non-specific binding was performed in parallel cultures incubated with 10/~M saralasin, and was typically less than 2% of the total binding. For determination of effect of treatments on affinity and number of AT 2 sites, cells were incubated with varying concentrations of angiotensin II and saturation analysis performed using the L I G A N D program [15]. Relative affinities of various angiotensin ligands was determined by varying the concentration of ligand in the presence of 30 pM [1251]Sarl,Ile8-angiotensin II. IC5o values were calculated by weighted nonlinear regression curve-fitting to the mass-action equation [7].
Results
Angiotensin H binding Binding of [125I]angiotensin II to intact R3T3 cells was saturable and of high affinity. Fig. 1 shows a representative saturation isotherm yielding an ap-
parent k d value of 0.6 nM and a Bmax of 125 ¢mol/ well. The pharmacology of angiotensin II binding to intact R3T3 cells is shown in Fig. 2. Angiotensin II, angiotensin III and saralasin all competed for binding of [125I]-Sarl,IleS-angiotensin II, yielding IC50 values of 3.54 nM, 1.11 nM and 3.36 nM, respectively. Angiotensin I also displaced the radioligand, yielding an IC50 value of 883 nM. The AT 2 specific ligands, PD 123319 and PD 123177 were able to completely displace the radiolabel, yielding IC50 values of 31.3 nM and 288 nM, while the AT 1 specific ligand, DuP 753 was unable to compete at concentrations up to 10 #M. This binding profile is essentially identical to that obtained in membranes prepared from R3T3 cells and shows that generation of AT 2 selective binding is not an artifact of membrane preparation. During the course of these studies, we realized that the amount of binding appeared to be dependent upon the length of time the cells had been in culture. Fig. 3 shows a growth curve for R3T3 cells with the corresponding number of AT 2 sites expressed per
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[Compound], nM Fig. 2. Pharmacologicalevaluationof~25I-Sar~,IleS-angiotensin II binding to whole R3T3 cells. Cells were incubated with 30 p M ~25I-Sar~,IleS-angiotensin II and the indicated concentrations of angiotensin I (@), angiotensin II (o), angiotensin III (&), saralasin (A), P D 123319 (m), P D 123177 (I--1) or D u P 753 ( x ) for 2 b at 37°C. Bound 12sI was determined as described in Materials and Methods.
202
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cell. These cells grow rapidly after plating and reach confluence in 3 - 4 days after which cell number is stable for several weeks. Interestingly, the number of AT2 sites expressed was very low in growing cells, and in fact it was difficult to demonstrate specific binding in sparse cultures. However, after cells reached confluence, there was a dramatic increase in
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Fig. 3. Effect of time in culture on expression of AT 2 sites. R3T3 cells were seeded into 12-well plates on day 0 at 2.104 cells/well. On indicated days, the number of AT 2 binding sites was determined as described in Materials and Methods. In parallel cultures, the number of cells/well was determined using a Coulter counter after removal of the cells by trypsin. For the experiment shown, there were three wells/condition and the error bars reflect the mean + S.E.M. of these wells. The experiment shown is representative of three separate experiments.
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Fig. 4. Effect of mitogens on expression of AT 2 sites. Confluent cultures of R3T3 cells were transferred to D M E M supplemented with 0.1 ~o B SA. After 24 h, cultures were stimulated with 10 fetal bovine serum (FBS), 1 #M bombesin (BN) or 100 ng/ml b F G F . Cultures were maintained for an additional 24 h, then the number of AT2 sites determined by binding analysis as described in Materials and Methods. The experiment shown is representative of three separate experiments.
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bFGF, ng/ml Fig. 5. Time and concentration dependence of b F G F treatment on AT= site expression. Confluent R3T3 cells were maintained in D M E M containing 0.1Yo BSA for 48 h prior to determination of the number of AT z binding sites. Cells were incubated with 100 ng/ml b F G F for the indicated times prior to assay (top panel), or with the indicated concentrations of b F G F during the final 24 h (lower panel). Shown is a representative of two separate experiments.
203 the n u m b e r o f A T 2 sites. This increase t e n d e d to stabilize a r o u n d 9 - 1 0 d a y s after plating. There was no change in the affinity o f angiotensin II for this binding site during the time in culture.
r e s p o n s e by 1 5 - 2 0 h. The r e s p o n s e to b F G F was also d o s e d e p e n d e n t , resulting in a h a l f - m a x i m u m effect at approx. 1 ng/ml b F G F .
Homologous up-regulation of A T2 binding sites Effect of mitogens on expression of A T 2 binding sites In o r d e r to further characterize the effect o f cell growth on expression o f A T 2 sites in cultured R3T3 cells, cells were p l a t e d a n d allowed to reach confluence, a n d then transferred to serum-free m e d i a for 48 h. During the final 24 h o f this treatment, cells were e x p o s e d to a variety o f agents that are mitogenic for R3T3 cells [7]. A s shown in Fig. 4, t r e a t m e n t o f cells with fetal bovine serum, b o m b e s i n or b F G F c a u s e d a large decrease in the n u m b e r o f cell-surface A T 2 binding sites. However, these treatments h a d no appreciable effect on the affinity o f the A T 2 sites for [125I]-angiotensin II ( d a t a n o t shown).
U s i n g a similar protocol, we n o t e d that treatment o f cells with ligands that b o u n d to A T u sites resulted in an increase in the n u m b e r o f these sites. This is illustrated in Fig. 6, which shows that treatment with angiotensin II, saralasin, P D 123319 or P D 123177 c a u s e d a 2 . 5 - 3 - f o l d increase in the n u m b e r o f cell4(X)
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A s the r e s p o n s e to b F G F was the largest, we further c h a r a c t e r i z e d its effect. Fig. 5 shows that treatm e n t with b F G F c a u s e d a detectable decrease in binding sites within a few hours, a n d a m a x i m u m
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Fig. 6. Effect of angiotensin ligands on AT2 site expression. R3T3 cells were maintained in DMEM containing 0.1% BSA for 48 h prior to determination of AT2 binding sites. Cells were incubated with 1 /~M angiotensin II (Ang II), saralasin (Sar), PD 123319 ('319), PD 123177 (' 177) or DuP 753 during the final 24 h. Shown is a representative of three separate experiments.
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Fig. 7. Time and concentration dependence of angiotensin ligand treatment on expression of AT2 sites. Confluent R3T3 cells were maintained in DMEM containing 0.1% BSA for 48 h prior to determination of the number of AT2 binding sites. Cells were incubated with 1 #M angiotensin II (O, Ang II), PD 123319 (V) or saralasin ( • ) for the indicated times prior to assay (top panel), or with the indicated concentrations of ligands during the final 24 h (lower panel). Shown is a representative of two separate experiments.
204
surface AT 2 sites. DuP753, which is not recognized by AT 2 sites failed to have an effect. As was noted above for mitogen effects, these treatments had no effect on the affinity of the AT 2 sites for [125I]angiotensin II. The increase in AT 2 sites elicited by these ligands was both time and dose-dependent as shown in Fig. 7. Response to either angiotensin, saralasin or PD 123319 tended to plateau by 24 h. The apparent EC50 value for this response was ~ 10 nM for angiotensin II and saralasin, and ~ 100 nM for PD 123319. While these values are somewhat higher than observed binding constants, the relative rank order is the same as for binding affinities. In order to investigate the mechanism of the upregulation, cells were treated with cycloheximide. Treatment of R3T3 cells with cycloheximide for 24 h resulted in a slight decrease in the number of AT 2 sites (Fig. 8). Cycloheximide treatment was also able to prevent the increase in AT 2 sites seen when cells were incubated with angiotensin II, saralasin or PD 123319. Under these conditions, incorporation of [3H]leucine into protein was inhibited by 80~o (data not shown). These results indicate that the mecha500000
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Fig. 8. Effect of cyeloheximide on ligand-mediated increases in AT2 binding sites. R3T3 cells were plated in multiwell plates and maintained in DMEM containing 0.1~o BSA for 48 h prior to determination of AT2 binding sites. During the final 24 h, cells were incubated with 1 #M angiotensin II (Ang II), saralasin (Sar) or PD 123319 ('319) either alone or in the presence of 1/zg/ml cycloheximide. Each point is an average of triplicate values and shown is a representative of two separate experiments.
nism by which AT 2 ligands increase AT 2 binding sites is dependent on protein synthesis.
Discussion We have previously shown that R3T3 fibroblasts express AT 2 binding sites for angiotensin II, but do not express the AT 1 angiotensin II receptor. Further, angiotensin II does not elicit any of the expected effects in these cells, such as stimulation of phosphatidylinositol turnover, effects on cyclic nucleotides, kinase activation or release of arachidonic acid metabolites. Additionally in these cells, the presence of AT 2 sites do not appear to modulate the activity of other receptor systems, as stimulation of second messenger production by a number of receptor systems is unchanged. Similarly, growth factor action is unchanged by AT2 ligands as these compounds exerted no effect on growth of R3T3 cells, or on growth stimulated by a number of mitogens [7]. Results reported here, however, show that while growth factor action may not be influenced by AT 2 sites, the level of AT 2 site expression is dramatically affected by growth factor activity. Exposure of R3T3 cells to a number of growth factors or growth stimulating conditions resulted in a rapid loss of cellsurface AT 2 binding activity. Conversely, attainment of a quiescent, confluent state resulted in expression of large amounts of AT 2 sites. This pattern of expression is reminiscent of that seen in the developing fetus. In that system, there is a large increase in expression of angiotensin binding sites in a number of tissues, primarily of mesenchyme origin (of which fibroblasts are derived), during fetal growth [14,1618]. Pharmacological analysis has recently shown that these sites are primarily AT 2 [14,17]. Subsequent to birth, there is a rapid decline in the expression of AT 2 sites [ 14,16]. It is unknown if endogenous growth factors in the fetal tissues are influencing expression of AT 2 sites, as observed here in R3T3 cells. In any event, the dramatic changes in levels of AT 2 sites suggests they serve a role in fetal develop-
205 ment and differentiation. Use of R3T3, and other culture models may provide insight into the mechanism by which these changes occur. The increase in angiotensin binding sites elicited by angiotensin ligands in R3T3 cells is intriguing, but not entirely unprecedented. In adrenal tissue, angiotensin II is well known for its ability to up-regulate its receptor [19]. Since this also results in an increased sensitivity to angiotensin, it is likely that this reflects an increase in AT 1 receptors. It will be interesting to determine if AT 2 sites are also upregulated in adrenal, as well as to determine if other angiotensin ligands mimic this ability of angiotensin. Up-regulation of angiotensin II receptors has also been noted in N1E-115 cells in response to differentiation and changes in intracellular cAMP and Ca 2 + [20]. Although it was not determined pharmacologically, there was an indication that not all of the observed increase in angiotensin binding activity could be accounted for by AT 1 receptors, suggesting an increase in AT 2 sites as well. While changes in cAMP and Ca 2 + occur during stimulation of R3T3 cells, and may lead to changes in expression of AT 2 sites, there are no observable changes in these parameters when cells are treated with angiotensin ligands. Yet, under these conditions, large changes in levels of AT 2 binding sites are apparent, suggesting other pathways are responsible for regulating levels of these binding sites. In R3T3 cells, it is tempting to speculate that ligand-induced up-regulation represents an agonist effect, and hence, a cellular response due to activation of AT 2 sites. This would imply that saralasin, PD 123319 and PD 123177 are all acting as agonists, with apparent efficacy as large as that for angiotensin II itself. An alternative explanation is that occupancy of AT 2 sites by ligand prevents its degradation. In the face of continuing synthesis, lack of degradation would result in accumulation of this site, and an apparent increase in the number of sites in response to ligands. Our experiments with cycloheximide do not support the latter possibility because AT 2 ligands were unable to overcome the block imposed by in-
hibition of protein synthesis. This suggests that the increase in sites observed when AT2 ligands are present is dependent on protein synthesis, and presumably mediated by ligand occupation of the AT 2 sites. Conclusive evidence will depend upon evaluation of AT 2 protein levels by an independent measure, and by evaluation of mRNA levels. Although no clear role may be currently assigned to AT 2 binding sites, it is apparent they are under the influence of a number of growth factor and regulatory conditions. In R3T3 cells, it appears that the communication is predominately one-way. For example, cell growth stimulated by b F G F is unaffected by treatment with angiotensin ligands, yet b F G F treatment leads to a rapid and substantial decrease of AT 2 binding activity. One possibility is that the AT 2 sites represented in R3T3 cells are not signaling components that acutely control the level of a second messenger, but are serving to sequester ligand, perhaps to prevent excessive activation of AT 1 receptors during fetal development, or to ensure a pool of active ligand during a critical phase of development. This level of sequestered ligand would then be dependent upon the local concentrations of growth factors and growth states of surrounding tissues. There are also suggestive data indicating that AT 2 sites may be heterogeneous [21,22], and perhaps be comprised of multiple components. In this regard, some AT 2 sites could be coupled to acute changes in a second messenger, while other sites might control longer term responses, or modulate responses mediated by other receptor systems. In any event, it is clear that levels of a binding site with high affinity for angiotensin peptides changes dramatically during development. Further research into this area is likely to reveal presently unappreciated roles for the family of angiotensin peptides.
Acknowledgement The authors wish to thank Dr. John Hodges for synthesis of DuP 753, PD 123319 and PD 123177.
206
References 1 Dzau, V.J. and Pratt, R.E., Renin-angiotensin system: biology, physiology and pharmacology. In H.A. Fozzard, E. Haber, R.B. Jennings, A.M. Katz and H.E. Morgan (Eds.), The Heart and Cardiovascular System, Raven Press, New York, 1986, pp. 1631-1662. 2 Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M.S. and Berstein, K.E., Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor, Nature, 351 (t991) 233-236. 3 Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J.J., Hasegawa, M., Matsuda, Y. and Inagami, T., Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor, Nature, 351 (1991) 230233. 4 Dudley, D.T., Panek, R.L., Major, T.C., Lu, G.H., Bruns, R.F., Klinkefus, B.A., Hodges, J.C. and Weishaar, R.E., Subclasses of angiotensin II binding sites and their functional significance, Mol. Pharm., 38 (1990) 370-377. 5 Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B., Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP 753 (All-1) and PD 123177 (AII-2), J. Pharm. Exp. Ther., 255 (1990) 584-592. 6 Whitebread, S., Mele, M., Kamber, B. and De Gasparo, M., Preliminary biochemical characterization of two angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 163 (1989) 284-291. 7 Dudley, D.T., Hubbell, S.E. and Summerfelt, R.M., Characterization of angiotensin II (ATe) binding sites in R3T3 cells, Mol. Pharm., 40 (1991) 360-367. 8 Pucell, A.G., Hodges, J.C., Sen, I., Bumpus, F.M. and Husain, A., Biochemical properties of the ovarian granulosa cell type 2-angiotensin II receptor, Endocrinology, 128 (1991) 1947-1959. 9 Sumners, C., Tang, W., Zelezna, B. and Raizada, M.K., Angiotensin II receptor subtypes are couple with distinct signaltransduction mechanisms in neurons and astrocytes from rat brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 7567-7571. 10 Bottari, S.P., King, I.N., Reichlin, S., Dahlstroem, I., Lydon, N. and De Gasparo, M., The angiotensin AT 2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase, Biochem. Biophys. Res. Commun., 183 (1992)206-211.
11 Webb, M.L., Liu, E.C.-K., Cohen, R.B., Hedberg, A., Bogosian, E.A., Monshizadegan, H., Molloy, C., Serafino, R., Moreland, S., Murphy, T.J. and Dickinson, K.E., Molecular characterization of angiotensin II type 2 receptors in rat pheochromocytoma cells, Peptides, 13 (1992) 499-508. 12 Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L.J., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B., Identification of angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 165 (1989) 196-203. 13 Tsutsumi, K. and Saavedra, J.M., Differential development of angiotensin II receptor subtypes in the rat brain, Endocrinology, 128 (1991) 630-632. 14 Grady, E.F., Sechi, L.A., Griffin, C.A., Schambelan, M. and Kalinyak, J.E., Expression of AT2 receptors in the developing rat fetus, J. Clin. Invest., 88 (1991) 921-933. 15 Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand binding systems, Anal. Biochem., 107 (1980) 220-229. 16 Millan, M.A., Carvallo, P., Izumi, S.-I., Zemel, S., Catt, K.J. and Aguilera, G., Novel sites of expression of functional angiotensin II receptors in the late gestation fetus, Science, 244 (1989) 1340-1342. 17 Viswanathan, M., Tsutsumi, K., Correa, F.M. and Saavedra, J.M., Changes in expression of angiotensin receptor subtypes in the rat aorta during development, Biochem. Biophys. Res. Commun., 179 (1991) 1361-1367. 18 Zemel, S., Millan, M.A., Feuillan, P. and Aguilera, G., Characterization and distribution of angiotensin-II receptors in the primate fetus, J. Clin. Endocrinol. Metab., 71 (1990) 10031007. 19 Aguilera, G., Schirar, A., Baukal, A. and Catt, K.J., Angiotensin II receptors. Properties and regulation in adrenal glomerulosa cells, Circ. Res., 46 (1980) 1-118-I-127. 20 Reagan, L.P., Xuehai, Y., Rubina, M., DePalo, L.R. and Fluharty, S.J., Up-regulation of angiotensin II receptors by in vitro differentiation of murine N 1E-115 neuroblastoma cells, Mol. Pharm., 38 (1990) 878-886. 21 Tsutsumi, K., Zorad, S. and Saavedra, J.M., The AT 2 subtype of the angiotensin II receptor has differential sensitivity to dithiothreitol in specific brain nuclei of young rats, Eur. J. Pharm., 226 (1992) 169-173. 22 Tsutsumi, K. and Saavedra, J.M., Heterogeneity of angiotensin II AT 2 receptors in the rat brain, Mol. Pharm., 41 (1992) 290-297.
199
REGPEP 01278
Regulated expression of angiotensin II (AT2) binding sites in R3T3 cells D a v i d T. D u d l e y and Rochelle M. Summerfelt Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI (USA) (Received 8 October 1992; accepted 11 December 1992)
Key words: PD 123319; Receptor; Growth factor; Mitogen; Fibroblast Summary R3T3 cells are a fibroblast cell line found to selectively express the AT 2 subtype of angiotensin II binding sites. We have previously shown that in these cells, the AT 2 sites do not appear to be coupled to G-proteins, do not modulate any of the common second messenger pathways associated with activation of angiotensin II receptors, and do not internalize bound ligand. Here we report that expression of AT 2 sites in these cells are subject to modulation by a variety of conditions. In actively growing cells the expression of AT 2 sites is very low, while in confluent, quiescent cells, the expression of AT 2 sites is markedly increased. Addition of serum, or growth factors, to quiescent cells causes a rapid decrease in the number of cell-surface AT 2 sites. Further, incubation of cells with ligands that bind to AT 2 sites causes a marked increase in the number of these sites in a time and dose dependent manner indicating homologous up-regulation of expression. These results indicate that expression of AT 2 sites in R3T3 cells is under sensitive and rapid control and further indicate that these cells may be an excellent model for studying the physiological regulation of expression of these sites.
Introduction Correspondence to: D.T. Dudley, Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, WarnerLambert Company, Ann Arbor, MI 48105, USA Abbreviations: bFGF, basic fibroblast growth factor; DMEM, Dulbeeco's Modified Essential Medium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; BSA, bovine serum alburnin; MEM, Minimal Essential Medium.
Angiotensin II is an eight amino acid peptide hormone known to have a variety of functions related to control of blood pressure and fluid homeostasis [ 1]. As a circulating hormone, many of its actions are thought to be mediated through cell-surface receptors. With the recent development of non-peptide
200
ligands that compete for binding with radiolabeled angiotensin II, it has become apparent that at least two major classes of binding sites or receptors exist for this peptide hormone. The AT 1 class, recognized by DuP 753 (also known as losartan), is commonly known as the angiotensin II receptor and cloning experiments have revealed it to be a member of the rhodopsin family of G-protein coupled receptors [2,3]. This receptor is typically coupled through phospholipase C and is responsible for essentially all of the currently known physiological actions of angiotensin II [4,5]. The AT 2 class is recognized by PD 123319 or CGP 42112A [4,6] and at present is not solely responsible for any known physiological actions of angiotensin II. Moreover, studies in cultured cells selectively expressing these sites have indicated that this site is not coupled through G-proteins and does not stimulate pathways typically associated with AT 1 receptors such as phosphatidylinositol turnover and inhibition of adenylate cyclase [7,8]. However, experiments in cultured neurons have revealed angiotensin II mediated decreases in cyclic G M P that are accounted for through AT 2 sites [9]. Recently, this effect has been attributed to changes in tyrosine phosphorylation, possibly through phosphatase activity [ 10]. While provocative, this has not been confirmed by other workers [11]. In adult animals, the AT 2 site is typically found in adrenal medulla [ 12], female reproductive organs [4] and certain brain regions [ 13]; areas that are largely distinct from where the AT 1 receptor is found. One of the most intriguing aspects of this site is its extensive localization in fetal tissue [ 14], and its rapid disappearance shortly after birth. We previously reported the finding of a line of fibroblast cells that selectively expressed AT 2 binding sites [7]. While these cells do not exhibit any detected perturbation in common signaling pathways upon exposure to angiotensin II, we report here that the expression of cell-surface AT 2 binding sites is dependent upon the growth state of the cells, and appears to be under rapid and sensitive control of
both mitogenic agents and ligands that bind to the site.
Materials and Methods
Materials [125I]Angiotensin II (2200 Ci/mmot) and [125I]Sarl,IleS-angiotensin II (2200 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Peptides were obtained from either Bachem Fine Chemicals (Torrance, CA), or Peninsula Laboratories (Belmont, CA). PD 123319, PD 123177 and DuP 753 were prepared as described [4]. Basic fibroblast growth factor (bFGF) was obtained from Intergen (Purchase, NY). Cell culture reagents were obtained from Gibco (Grand Island, NY) and fetal bovine serum from HyClone Laboratories (Logan, UT). Cell culture R3T3 cells, a mouse fibroblast line, were cultured as described [7] using Dulbecco's modified essential medium supplemented with 10~o fetal bovine serum. All experiments used cells grown as a monolayer. Cells were enumerated using a Coulter counter after release from plastic substrates with trypsin. Angiotensin H binding assay For most experiments, R3T3 cells were grown in 12-well plates and treated as indicated. Following treatment, cells were washed twice with 0.5 ml phosphate-buffered saline and incubated at 4°C for 3 rain in acid-glycine buffer (150 mM NaC1, 50 mM glycine, 1~o BSA, pH 3) in order to remove any ligands bound to cell-surface AT 2 binding sites. Cells were then rewashed twice with phosphate-buffered saline and incubated with minimal essential medium (MEM) supplemented with 20 mM Hepes, 0.1 ~o BSA, 0.2~o bacitracin and [125I]angiotensin II for 2 h at 37°C. Cells were then washed twice with icecold phosphate-buffered saline, solubilized in 2~o sodium dodecyl sulfate and the solubilized material
201
counted for 7 radioactivity. Non-specific binding was performed in parallel cultures incubated with 10/~M saralasin, and was typically less than 2% of the total binding. For determination of effect of treatments on affinity and number of AT 2 sites, cells were incubated with varying concentrations of angiotensin II and saturation analysis performed using the L I G A N D program [15]. Relative affinities of various angiotensin ligands was determined by varying the concentration of ligand in the presence of 30 pM [1251]Sarl,Ile8-angiotensin II. IC5o values were calculated by weighted nonlinear regression curve-fitting to the mass-action equation [7].
Results
Angiotensin H binding Binding of [125I]angiotensin II to intact R3T3 cells was saturable and of high affinity. Fig. 1 shows a representative saturation isotherm yielding an ap-
parent k d value of 0.6 nM and a Bmax of 125 ¢mol/ well. The pharmacology of angiotensin II binding to intact R3T3 cells is shown in Fig. 2. Angiotensin II, angiotensin III and saralasin all competed for binding of [125I]-Sarl,IleS-angiotensin II, yielding IC50 values of 3.54 nM, 1.11 nM and 3.36 nM, respectively. Angiotensin I also displaced the radioligand, yielding an IC50 value of 883 nM. The AT 2 specific ligands, PD 123319 and PD 123177 were able to completely displace the radiolabel, yielding IC50 values of 31.3 nM and 288 nM, while the AT 1 specific ligand, DuP 753 was unable to compete at concentrations up to 10 #M. This binding profile is essentially identical to that obtained in membranes prepared from R3T3 cells and shows that generation of AT 2 selective binding is not an artifact of membrane preparation. During the course of these studies, we realized that the amount of binding appeared to be dependent upon the length of time the cells had been in culture. Fig. 3 shows a growth curve for R3T3 cells with the corresponding number of AT 2 sites expressed per
125
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0. l
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[Compound], nM Fig. 2. Pharmacologicalevaluationof~25I-Sar~,IleS-angiotensin II binding to whole R3T3 cells. Cells were incubated with 30 p M ~25I-Sar~,IleS-angiotensin II and the indicated concentrations of angiotensin I (@), angiotensin II (o), angiotensin III (&), saralasin (A), P D 123319 (m), P D 123177 (I--1) or D u P 753 ( x ) for 2 b at 37°C. Bound 12sI was determined as described in Materials and Methods.
202
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cell. These cells grow rapidly after plating and reach confluence in 3 - 4 days after which cell number is stable for several weeks. Interestingly, the number of AT2 sites expressed was very low in growing cells, and in fact it was difficult to demonstrate specific binding in sparse cultures. However, after cells reached confluence, there was a dramatic increase in
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Fig. 3. Effect of time in culture on expression of AT 2 sites. R3T3 cells were seeded into 12-well plates on day 0 at 2.104 cells/well. On indicated days, the number of AT 2 binding sites was determined as described in Materials and Methods. In parallel cultures, the number of cells/well was determined using a Coulter counter after removal of the cells by trypsin. For the experiment shown, there were three wells/condition and the error bars reflect the mean + S.E.M. of these wells. The experiment shown is representative of three separate experiments.
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Fig. 4. Effect of mitogens on expression of AT 2 sites. Confluent cultures of R3T3 cells were transferred to D M E M supplemented with 0.1 ~o B SA. After 24 h, cultures were stimulated with 10 fetal bovine serum (FBS), 1 #M bombesin (BN) or 100 ng/ml b F G F . Cultures were maintained for an additional 24 h, then the number of AT2 sites determined by binding analysis as described in Materials and Methods. The experiment shown is representative of three separate experiments.
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bFGF, ng/ml Fig. 5. Time and concentration dependence of b F G F treatment on AT= site expression. Confluent R3T3 cells were maintained in D M E M containing 0.1Yo BSA for 48 h prior to determination of the number of AT z binding sites. Cells were incubated with 100 ng/ml b F G F for the indicated times prior to assay (top panel), or with the indicated concentrations of b F G F during the final 24 h (lower panel). Shown is a representative of two separate experiments.
203 the n u m b e r o f A T 2 sites. This increase t e n d e d to stabilize a r o u n d 9 - 1 0 d a y s after plating. There was no change in the affinity o f angiotensin II for this binding site during the time in culture.
r e s p o n s e by 1 5 - 2 0 h. The r e s p o n s e to b F G F was also d o s e d e p e n d e n t , resulting in a h a l f - m a x i m u m effect at approx. 1 ng/ml b F G F .
Homologous up-regulation of A T2 binding sites Effect of mitogens on expression of A T 2 binding sites In o r d e r to further characterize the effect o f cell growth on expression o f A T 2 sites in cultured R3T3 cells, cells were p l a t e d a n d allowed to reach confluence, a n d then transferred to serum-free m e d i a for 48 h. During the final 24 h o f this treatment, cells were e x p o s e d to a variety o f agents that are mitogenic for R3T3 cells [7]. A s shown in Fig. 4, t r e a t m e n t o f cells with fetal bovine serum, b o m b e s i n or b F G F c a u s e d a large decrease in the n u m b e r o f cell-surface A T 2 binding sites. However, these treatments h a d no appreciable effect on the affinity o f the A T 2 sites for [125I]-angiotensin II ( d a t a n o t shown).
U s i n g a similar protocol, we n o t e d that treatment o f cells with ligands that b o u n d to A T u sites resulted in an increase in the n u m b e r o f these sites. This is illustrated in Fig. 6, which shows that treatment with angiotensin II, saralasin, P D 123319 or P D 123177 c a u s e d a 2 . 5 - 3 - f o l d increase in the n u m b e r o f cell4(X)
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A s the r e s p o n s e to b F G F was the largest, we further c h a r a c t e r i z e d its effect. Fig. 5 shows that treatm e n t with b F G F c a u s e d a detectable decrease in binding sites within a few hours, a n d a m a x i m u m
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Fig. 6. Effect of angiotensin ligands on AT2 site expression. R3T3 cells were maintained in DMEM containing 0.1% BSA for 48 h prior to determination of AT2 binding sites. Cells were incubated with 1 /~M angiotensin II (Ang II), saralasin (Sar), PD 123319 ('319), PD 123177 (' 177) or DuP 753 during the final 24 h. Shown is a representative of three separate experiments.
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Fig. 7. Time and concentration dependence of angiotensin ligand treatment on expression of AT2 sites. Confluent R3T3 cells were maintained in DMEM containing 0.1% BSA for 48 h prior to determination of the number of AT2 binding sites. Cells were incubated with 1 #M angiotensin II (O, Ang II), PD 123319 (V) or saralasin ( • ) for the indicated times prior to assay (top panel), or with the indicated concentrations of ligands during the final 24 h (lower panel). Shown is a representative of two separate experiments.
204
surface AT 2 sites. DuP753, which is not recognized by AT 2 sites failed to have an effect. As was noted above for mitogen effects, these treatments had no effect on the affinity of the AT 2 sites for [125I]angiotensin II. The increase in AT 2 sites elicited by these ligands was both time and dose-dependent as shown in Fig. 7. Response to either angiotensin, saralasin or PD 123319 tended to plateau by 24 h. The apparent EC50 value for this response was ~ 10 nM for angiotensin II and saralasin, and ~ 100 nM for PD 123319. While these values are somewhat higher than observed binding constants, the relative rank order is the same as for binding affinities. In order to investigate the mechanism of the upregulation, cells were treated with cycloheximide. Treatment of R3T3 cells with cycloheximide for 24 h resulted in a slight decrease in the number of AT 2 sites (Fig. 8). Cycloheximide treatment was also able to prevent the increase in AT 2 sites seen when cells were incubated with angiotensin II, saralasin or PD 123319. Under these conditions, incorporation of [3H]leucine into protein was inhibited by 80~o (data not shown). These results indicate that the mecha500000
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Fig. 8. Effect of cyeloheximide on ligand-mediated increases in AT2 binding sites. R3T3 cells were plated in multiwell plates and maintained in DMEM containing 0.1~o BSA for 48 h prior to determination of AT2 binding sites. During the final 24 h, cells were incubated with 1 #M angiotensin II (Ang II), saralasin (Sar) or PD 123319 ('319) either alone or in the presence of 1/zg/ml cycloheximide. Each point is an average of triplicate values and shown is a representative of two separate experiments.
nism by which AT 2 ligands increase AT 2 binding sites is dependent on protein synthesis.
Discussion We have previously shown that R3T3 fibroblasts express AT 2 binding sites for angiotensin II, but do not express the AT 1 angiotensin II receptor. Further, angiotensin II does not elicit any of the expected effects in these cells, such as stimulation of phosphatidylinositol turnover, effects on cyclic nucleotides, kinase activation or release of arachidonic acid metabolites. Additionally in these cells, the presence of AT 2 sites do not appear to modulate the activity of other receptor systems, as stimulation of second messenger production by a number of receptor systems is unchanged. Similarly, growth factor action is unchanged by AT2 ligands as these compounds exerted no effect on growth of R3T3 cells, or on growth stimulated by a number of mitogens [7]. Results reported here, however, show that while growth factor action may not be influenced by AT 2 sites, the level of AT 2 site expression is dramatically affected by growth factor activity. Exposure of R3T3 cells to a number of growth factors or growth stimulating conditions resulted in a rapid loss of cellsurface AT 2 binding activity. Conversely, attainment of a quiescent, confluent state resulted in expression of large amounts of AT 2 sites. This pattern of expression is reminiscent of that seen in the developing fetus. In that system, there is a large increase in expression of angiotensin binding sites in a number of tissues, primarily of mesenchyme origin (of which fibroblasts are derived), during fetal growth [14,1618]. Pharmacological analysis has recently shown that these sites are primarily AT 2 [14,17]. Subsequent to birth, there is a rapid decline in the expression of AT 2 sites [ 14,16]. It is unknown if endogenous growth factors in the fetal tissues are influencing expression of AT 2 sites, as observed here in R3T3 cells. In any event, the dramatic changes in levels of AT 2 sites suggests they serve a role in fetal develop-
205 ment and differentiation. Use of R3T3, and other culture models may provide insight into the mechanism by which these changes occur. The increase in angiotensin binding sites elicited by angiotensin ligands in R3T3 cells is intriguing, but not entirely unprecedented. In adrenal tissue, angiotensin II is well known for its ability to up-regulate its receptor [19]. Since this also results in an increased sensitivity to angiotensin, it is likely that this reflects an increase in AT 1 receptors. It will be interesting to determine if AT 2 sites are also upregulated in adrenal, as well as to determine if other angiotensin ligands mimic this ability of angiotensin. Up-regulation of angiotensin II receptors has also been noted in N1E-115 cells in response to differentiation and changes in intracellular cAMP and Ca 2 + [20]. Although it was not determined pharmacologically, there was an indication that not all of the observed increase in angiotensin binding activity could be accounted for by AT 1 receptors, suggesting an increase in AT 2 sites as well. While changes in cAMP and Ca 2 + occur during stimulation of R3T3 cells, and may lead to changes in expression of AT 2 sites, there are no observable changes in these parameters when cells are treated with angiotensin ligands. Yet, under these conditions, large changes in levels of AT 2 binding sites are apparent, suggesting other pathways are responsible for regulating levels of these binding sites. In R3T3 cells, it is tempting to speculate that ligand-induced up-regulation represents an agonist effect, and hence, a cellular response due to activation of AT 2 sites. This would imply that saralasin, PD 123319 and PD 123177 are all acting as agonists, with apparent efficacy as large as that for angiotensin II itself. An alternative explanation is that occupancy of AT 2 sites by ligand prevents its degradation. In the face of continuing synthesis, lack of degradation would result in accumulation of this site, and an apparent increase in the number of sites in response to ligands. Our experiments with cycloheximide do not support the latter possibility because AT 2 ligands were unable to overcome the block imposed by in-
hibition of protein synthesis. This suggests that the increase in sites observed when AT2 ligands are present is dependent on protein synthesis, and presumably mediated by ligand occupation of the AT 2 sites. Conclusive evidence will depend upon evaluation of AT 2 protein levels by an independent measure, and by evaluation of mRNA levels. Although no clear role may be currently assigned to AT 2 binding sites, it is apparent they are under the influence of a number of growth factor and regulatory conditions. In R3T3 cells, it appears that the communication is predominately one-way. For example, cell growth stimulated by b F G F is unaffected by treatment with angiotensin ligands, yet b F G F treatment leads to a rapid and substantial decrease of AT 2 binding activity. One possibility is that the AT 2 sites represented in R3T3 cells are not signaling components that acutely control the level of a second messenger, but are serving to sequester ligand, perhaps to prevent excessive activation of AT 1 receptors during fetal development, or to ensure a pool of active ligand during a critical phase of development. This level of sequestered ligand would then be dependent upon the local concentrations of growth factors and growth states of surrounding tissues. There are also suggestive data indicating that AT 2 sites may be heterogeneous [21,22], and perhaps be comprised of multiple components. In this regard, some AT 2 sites could be coupled to acute changes in a second messenger, while other sites might control longer term responses, or modulate responses mediated by other receptor systems. In any event, it is clear that levels of a binding site with high affinity for angiotensin peptides changes dramatically during development. Further research into this area is likely to reveal presently unappreciated roles for the family of angiotensin peptides.
Acknowledgement The authors wish to thank Dr. John Hodges for synthesis of DuP 753, PD 123319 and PD 123177.
206
References 1 Dzau, V.J. and Pratt, R.E., Renin-angiotensin system: biology, physiology and pharmacology. In H.A. Fozzard, E. Haber, R.B. Jennings, A.M. Katz and H.E. Morgan (Eds.), The Heart and Cardiovascular System, Raven Press, New York, 1986, pp. 1631-1662. 2 Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M.S. and Berstein, K.E., Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor, Nature, 351 (t991) 233-236. 3 Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J.J., Hasegawa, M., Matsuda, Y. and Inagami, T., Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor, Nature, 351 (1991) 230233. 4 Dudley, D.T., Panek, R.L., Major, T.C., Lu, G.H., Bruns, R.F., Klinkefus, B.A., Hodges, J.C. and Weishaar, R.E., Subclasses of angiotensin II binding sites and their functional significance, Mol. Pharm., 38 (1990) 370-377. 5 Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B., Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP 753 (All-1) and PD 123177 (AII-2), J. Pharm. Exp. Ther., 255 (1990) 584-592. 6 Whitebread, S., Mele, M., Kamber, B. and De Gasparo, M., Preliminary biochemical characterization of two angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 163 (1989) 284-291. 7 Dudley, D.T., Hubbell, S.E. and Summerfelt, R.M., Characterization of angiotensin II (ATe) binding sites in R3T3 cells, Mol. Pharm., 40 (1991) 360-367. 8 Pucell, A.G., Hodges, J.C., Sen, I., Bumpus, F.M. and Husain, A., Biochemical properties of the ovarian granulosa cell type 2-angiotensin II receptor, Endocrinology, 128 (1991) 1947-1959. 9 Sumners, C., Tang, W., Zelezna, B. and Raizada, M.K., Angiotensin II receptor subtypes are couple with distinct signaltransduction mechanisms in neurons and astrocytes from rat brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 7567-7571. 10 Bottari, S.P., King, I.N., Reichlin, S., Dahlstroem, I., Lydon, N. and De Gasparo, M., The angiotensin AT 2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase, Biochem. Biophys. Res. Commun., 183 (1992)206-211.
11 Webb, M.L., Liu, E.C.-K., Cohen, R.B., Hedberg, A., Bogosian, E.A., Monshizadegan, H., Molloy, C., Serafino, R., Moreland, S., Murphy, T.J. and Dickinson, K.E., Molecular characterization of angiotensin II type 2 receptors in rat pheochromocytoma cells, Peptides, 13 (1992) 499-508. 12 Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L.J., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B., Identification of angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 165 (1989) 196-203. 13 Tsutsumi, K. and Saavedra, J.M., Differential development of angiotensin II receptor subtypes in the rat brain, Endocrinology, 128 (1991) 630-632. 14 Grady, E.F., Sechi, L.A., Griffin, C.A., Schambelan, M. and Kalinyak, J.E., Expression of AT2 receptors in the developing rat fetus, J. Clin. Invest., 88 (1991) 921-933. 15 Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand binding systems, Anal. Biochem., 107 (1980) 220-229. 16 Millan, M.A., Carvallo, P., Izumi, S.-I., Zemel, S., Catt, K.J. and Aguilera, G., Novel sites of expression of functional angiotensin II receptors in the late gestation fetus, Science, 244 (1989) 1340-1342. 17 Viswanathan, M., Tsutsumi, K., Correa, F.M. and Saavedra, J.M., Changes in expression of angiotensin receptor subtypes in the rat aorta during development, Biochem. Biophys. Res. Commun., 179 (1991) 1361-1367. 18 Zemel, S., Millan, M.A., Feuillan, P. and Aguilera, G., Characterization and distribution of angiotensin-II receptors in the primate fetus, J. Clin. Endocrinol. Metab., 71 (1990) 10031007. 19 Aguilera, G., Schirar, A., Baukal, A. and Catt, K.J., Angiotensin II receptors. Properties and regulation in adrenal glomerulosa cells, Circ. Res., 46 (1980) 1-118-I-127. 20 Reagan, L.P., Xuehai, Y., Rubina, M., DePalo, L.R. and Fluharty, S.J., Up-regulation of angiotensin II receptors by in vitro differentiation of murine N 1E-115 neuroblastoma cells, Mol. Pharm., 38 (1990) 878-886. 21 Tsutsumi, K., Zorad, S. and Saavedra, J.M., The AT 2 subtype of the angiotensin II receptor has differential sensitivity to dithiothreitol in specific brain nuclei of young rats, Eur. J. Pharm., 226 (1992) 169-173. 22 Tsutsumi, K. and Saavedra, J.M., Heterogeneity of angiotensin II AT 2 receptors in the rat brain, Mol. Pharm., 41 (1992) 290-297.