Regulation of Type 1 Angiotensin II Receptor Messenger Ribonucleic Acid Expression in Human Adrenocortical Carcinoma H295 Cells*
IAN M. BIRDt, J. IAN MASON, AND WILLIAM E. RAINEY
Departments of Obstetrics and Gynecology and Biochemistry and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235
ABSTRACT
We have studied the hormonal regulation of type 1 angiotensin-II receptor (AT1-R) mRNA expression and [125I]angiotensin-II ([125]]AII) binding in human adrenocortical carcinoma H295 cells, which exhibit predominantly AT1-subtype receptors. Activation of the cAMP signal- ing pathway with forskolin or (Bu)2CAMP caused a rapid decrease in AT1-R mRNA levels (decreased 65% within 3 h). This preceded a time- dependent (maximal, 70% within 12 h) and dose-dependent (IC50, 2 MM forskolin) loss of [125]]AII binding together with decreased phosphoi- nositidase-C activation (72% decrease) on subsequent AII challenge. Thus, the decreases in AT1-R mRNA levels and functional receptor expression parallel each other in response to activation of protein
kinase-A. AII treatment also caused a rapid loss in AT1-R mRNA (maximal, 80% decrease within 3 h), but 48-h treatment caused both [125]]AII binding and the subsequent phosphoinositidase-C response to decrease by only 6% (P < 0.05) and 22% (P < 0.05), respectively. The effect of AII on AT1-R mRNA levels was fully reproduced by the combination of calcium ionophore (A23187) and phorbol ester (12-0- tetradecanoylphorbol 13-acetate), suggesting that AII action was through protein kinase-C and possibly other Ca2+-sensitive protein kinases. The effect of AII, but not forskolin, was reversed by treatment in the presence of cycloheximide. In conclusion, control of AT1-R expression is differentially regulated by adenylate cyclase and phos- phoinositidase-C signaling pathways, which act at multiple levels in human adrenocortical cells. (Endocrinology 134: 2468-2474, 1994)
T HE RENIN -angiotensin system plays a key role in reg- ulating blood pressure, fluid, and electrolyte homeosta- sis. The active hormone in this system is angiotensin-II (AII), which exerts a diversity of actions in multiple target tissues, including the adrenal, liver, smooth muscle, heart, brain, and kidney. In the adrenal cortex, AII acutely regulates aldoster- one production (1, 2) and chronically regulates the expression of several enzymes involved in aldosterone biosynthesis from the zona glomerulosa (3).
The recent development of novel nonpeptide All antago- nists has unequivocally demonstrated the existence of two distinct subtypes of angiotensin-binding sites. These AII receptor subgroups have been designated type 1 (AT1-R) and type 2 (AT2-R) (4). Those receptors involved in the control of vasoconstriction and electrolyte homeostasis are of the AT1 subtype, functionally coupled to phosphoinositidase-C. The cDNA encoding the rat, bovine, and human AT1-R as well as the human genomic sequence have been isolated (5- 9). These sequences show a very high degree of homology in the protein-coding region, and the predicted amino acid sequence includes seven membrane-spanning «-helical do- mains.
Received October 27, 1993.
Address all correspondence and requests for reprints to: Dr. William E. Rainey, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dal- las, Texas 75235-9032.
* This work was supported in part by NIH, DHHS, Grants AG-08175 and HD-11149 (to J.I.M.) and American Heart Association Texas Affiliate Grant 93R-082 and a grant from Merck Research Laboratories (to W.E.R.).
t Supported in part by NIH Training Grant T32-HD-07190.
Little is known concerning the mechanisms controlling the regulation of expression of the AT1-R. The rat, but not bovine or human, AT1-R exists in two subforms, which encode 95% identical amino acid sequences and exhibit similar pharma- cology, but also have different 5’-untranslated sequences and show differential expression in various tissues (10). Treatment of rats with a low sodium diet increases AT1B-R mRNA in the rat adrenal, whereas the AT1-R antagonist, DuP753, appears to decrease AT1B-R mRNA levels. A recent study has also indicated that this effect is due not to hyper- plasia of the adrenal zona glomerulosa, but to increased mRNA per zona glomerulosa cell (11). The only information on hormonal regulation of AT1-R expression in the human adrenal comes from a recent study on primary cultures of cortisol-secreting zona fasciculata/reticularis cells from the human adrenal cortex (12). In contrast to the findings in rat adrenal, these results suggested that AII may decrease AT1- R mRNA levels. No data were presented comparing the down-regulation of mRNA and the level of AII binding. In the present study using human adrenocortical carcinoma H295 cells, which express functional AT1-R at a high level (13), we examined the regulation of AT1-R mRNA levels and AT1-R binding in response to activators of both the adenylate cyclase and phosphoinositidase-C pathways. Our results in- dicate that both protein kinase-A and the calcium/protein kinase-C signaling pathways are capable of rapidly down- regulating levels of AT1-R mRNA, but have differential ef- fects on AT1-R-binding sites. Our findings demonstrate that regulation of AT1-R expression is more complex than previ- ously described and is mediated through multiple signaling pathways acting at many levels.
Materials and Methods
Unless otherwise stated, materials are as reported previously (13). The agonists and antagonists used were A-II(Asp1-Ile5), 12-O-tetrade- canoylphorbol 13-acetate (TPA), A23187, forskolin, (Bu)2CAMP (all from Sigma Chemical Co., St. Louis, MO), DuP753 (Losartan, DuPont, Wil- mington, DE), and PD123319 (Parke-Davis, Ann Arbor, MI).
Cell culture
NCI-H295 cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD), were maintained in a 1:1 mixture of Dulbecco’s Modified Eagle’s and Ham’s F-12 media (DME/F12 containing pyridox- ine HCI, L-glutamine, and 15 mm HEPES; catalog no. 11331-014, Gibco- BRL Gaithersburg, MD) supplemented with insulin (6.25 µg/ml), trans- ferrin (6.25 ug/ml), selenium (6.25 ng/ml), linoleic acid (5.35 µg/ml; 1% ITS Plus, Collaborative Research, Bedford, MA), 2% low protein serum replacement-1 (LPSR-1, Sigma), and antibiotics. Cells were maintained and grown on 75-cm2 flasks at 37 C under an atmosphere of 5% CO2- 95% air. During the initial 3 months of culture, only attached cells were retained when medium was changed. This allowed the isolation of a population of H295 cells which grew as a monolayer culture rather than as a suspension (13, 14). This population of cells has been deposited with the ATCC and are designated H295-R. The studies originating from our laboratory were carried out using these cells. Cell monolayers were subcultured, and after 48 h, medium were removed and replaced with serum-free medium (DME/F12 containing antibiotics and 0.01% BSA). Cells were cultured for a further 24 h, then rinsed and treated in the same medium.
Receptor binding assay
AII receptor binding was determined using conditions described previously (13). [125I]AII (2000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Receptor assays were performed on 12-well plates containing approximately 0.2 × 106 cells/well. Binding was carried out for 1 h at 37 C in 0.5 ml binding medium (DME/F12, 0.5% BSA, 0.1% bacitracin, and 15 mm HEPES, pH 7.4) with 200,000 cpm [125]]AII in the presence of 5 X 10-10 M unlabeled AII and 10 AM PD123319. At the end of the incubation, cells were rinsed three times with DME/F12 medium at 4 C. The cell layer was then lysed in NaOH (0.5 M) containing sodium deoxycholate (0.4%), and the radioactivity associated with the lysates was counted in a y-counter. Protein determinations at this time demonstrated that none of the treatments altered cellular protein per well by more than 15%.
Preparation of AT1-R cDNA and generation of [32P]probe
Oligonucleotides used in the generation of probe were based on the published sequence for the bovine adrenal AT1-R cDNA (15). Oligonu- cleotide BAT,F (AGA ATC CAA GAT GAT TGT CCC A) corresponded to bases 37-58 inclusive of the protein-coding region, whereas oligo BATIR (GGC CTT TGG GGG AAT GTA TTT CAG) was the comple- mentary sequence to bases 949-972 inclusive. Total RNA isolated from bovine adrenocortical cell cultures was used for reverse transcription- polymerase chain reaction generation of the AT1-R cDNA using BAT,F and BATIR. A product of about 950 bases was obtained, as expected, corresponding to bases 37-972 of the protein-coding sequence. The PCR product was ligated into the pCR1000 (InVitrogen, San Diego, CA) vector and used to transform competent Escherichia coli. Positive colonies were identified by hybridization to 32P-labeled PCR product and con- firmed by digestion of plasmid using restriction enzymes HindIII and EcoRI to yield a 1-kilobase fragment. Positive clone (BAT156) was sequenced from each end using the dideoxynucleotide termination method. A sequence identical to that previously reported (15) was obtained. The plasmid was used as template to generate antisense probes for Northern analysis by asymmetric PCR amplification (BAT,R:BATIF ratio = 100:1) in the presence of [32P]deoxy-CTP, giving incorporation of label in excess of 75%.
Northern analysis for AT1-R mRNA
Cells on 100-mm culture dishes were lysed at 4 C into 1 ml RNAzol- B solution (Cinna Biotecx, Houston, TX) and transferred to a microfuge tube. Phase separation was achieved by mixing with 0.15 ml CHCI3, incubation at 4 C for 5 min, and centrifugation (12,000 × g; 20 min; 4 C). The upper phase (0.7 ml) was transferred to a second microfuge tube, and RNA was then precipitated by the addition of 0.8 ml isopro- panol and standing for 1 h at -20 C. RNA was recovered by centrifu- gation (30 min; 12,000 × g; 4 C), and the recovered pellet was washed once in 75% ethanol (1.0 ml) before drying under air and dissolving in 1 mm EDTA, pH 7.0 (0.1 ml). After determination of recovery and purity by measuring absorbance at 260 and 280 nm, samples were precipitated by the addition of 1 ml absolute ethanol and 0.01 ml sodium acetate (3 M; pH 5.2) and stored at -70 C before analysis.
Samples were separated by electrophoresis on gels containing 1.1% agarose in the presence of formaldehyde. The presence and integrity of the major RNA species were examined under UV light to ensure con- sistency between lanes. RNA was transferred to a Magna NT membrane (MSI) by pressure blotting (75 psi; 1 h; PossiBlot Pressure Blotter, Stratagene, La Jolla, CA) and cross-linked under UV light. Prehybridi- zation was carried out at 42 C overnight in a final buffer composition of 50% formamide, 5 x SSC, 1 x PE, and 50 ug/ml transfer RNA [20 × SSC contains 3.0 M NaCl and 0.3 M trisodium citrate, pH 7.0; 5 x PE contains 250 mm Tris-HCI (pH 7.5), 0.5% sodium pyrophosphate, 5% sodium dodecyl sulfate (SDS), 1% polyvinylpyrrolidone, 1% Ficoll, 25 mM EDTA, and 1% BSA]. Hybridizations were performed in the same buffer at 42 C for 16-24 h, using bovine AT1-R antisense probe (labeled by asymmetric PCR with [32P]deoxy-CTP; Amersham). The blots were then washed in 2 x SSC containing 0.1% SDS at room temperature for 15 min and in 0.1 x SSC containing 0.1% SDS at room temperature twice for 30 min each time before drying and exposure to film (Hyper- film, Amersham). Blots were subsequently stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA. Anti- sense probe was prepared by asymmetric PCR amplification of the human cDNA (bases 39-900) in the presence of [32P]deoxy-CTP, and hybridization and posthybridization wash conditions were exactly as described above.
Studies of phosphoinositidase-C activation
Cells cultured on 24-well plates were prelabeled for 48 h in DME/ F12-0.01% BSA medium supplemented with 10 „Ci/ml [3H]inositol (Amersham). Before subsequent acute treatment with AII, cells were rinsed once in DME/F12 medium and incubated for 30 min in DME/ F12 containing 10 mm LiCl (to inhibit phosphoinositol phosphatases). Cells were then treated in a final volume of 0.5 ml with or without AIl (100 nm) for 30 min. At the end of this time, 0.25 ml ice-cold perchloric acid (15%) was added to each well to denature proteins and lyse the cells. The base of each well was then scraped, and the well contents were recovered, with a 0.5-ml water wash, into a microfuge tube (1.5 ml). Precipitated material was pelleted by centrifugation (12,000 × g; 2 min), and the supernatant was transferred to a glass tube. The aqueous phase (containing inositol and phosphoinositols) was then neutralized by mixing with freon-octylamine.
Separation of [3H]inositol from combined [3H]inositol phosphates was carried out on columns of AG1X8 anion exchange resin. Samples were loaded onto individual columns (0.25 ml resin/column), and all unbound [3H]inositol was eluted with water (twice, 4 ml each time). Columns were then eluted with 1 M ammonium formate-0.1 M formic acid (twice, 2 ml each time) to recover the bound [3H]inositol phosphates (up to inositol tetrakisphosphate). The radioactivity of the recovered [3H]ino- sitol phosphates fraction was then determined by liquid scintillation counting for 5 min each, or less if a counting error of 1% outside 2 SD (to 40,000 counts) could be achieved in that time. All values shown are corrected for volume.
Statistical analysis
Statistical analysis was accomplished using analysis of variance, followed by Student-Newman-Keuls multiple comparison analysis.
Results
The effects of treatment of H295 cells for 24 h with an activator of phosphoinositidase-C (AII) or with the potent activator of adenylate cyclase (forskolin) resulted in a marked reduction in AT1-R mRNA levels (Fig. 1). Treatment with TPA reduced the level of AT1-R mRNA, but failed to fully reproduce the effect of AII. Treatment with (Bu)2CAMP fully reproduced the effect of forskolin (data not shown), whereas ACTH had little effect (Fig. 1), consistent with the low expression of ACTH receptors in these cells.
When changes in AII binding to AT1-R were examined (Fig. 2a), 48-h treatment with forskolin and dbcAMP was found to decrease binding 60-70%, consistent with their effects on AT1-R mRNA. The effects of forskolin and (Bu)2CAMP on AT1-R binding were also found to be concen- tration dependent (Fig. 2b). Once again, ACTH had little or no effect on binding, probably due to its weak agonist effects in these cells. AII treatment caused only a small (6%; P < 0.05) decrease in binding (Fig. 2a), in contrast to its marked effects on levels of AT1-R mRNA (Fig. 1).
In addition to studying changes in AII binding, we exam- ined alterations in the phosphoinositidase-C response to AII after pretreatment with AII or forskolin. Cells pretreated with forskolin showed a marked attenuation (72%) of the
Con
A-II
TPA
ACTH
Forsk
9.5
7.5
- 28S
4.4
AT,-R
2.4
- 18S
1.4
- 28S
- G3PDH
a)
120
100
125|-AII BOUND (% of control cells)
80
60
40
20
0
Control
All
ACTH
dbcAMP
Forskolin
b)
120
100
125|-All BOUND (% of control cells)
2
80
60
40
ACTH
20
Forskolin
dbcAMP
0
Control
11-
10
9
8
7
à
5
4
3
8
2
Log[Agonist](M)
subsequent AII-stimulated increase in phosphoinositol ac- cumulation, whereas cells pretreated with All had only a marginally attenuated (22%; P < 0.05) response (Table 1). Neither AII nor forskolin pretreatment had any effect on phosphoinositide prelabeling with [3H]inositol, indicating that the effects of these agonists on phoshoinositol accumu- lation were not due to differences in steady state labeling of the phosphoinositides. Thus, H295 cells do not possess spare receptors to AII, so changes in binding were sufficient to result in altered phosphoinositidase-C activation in these cells.
Examination of the time course of the effects of forskolin revealed that a half-maximal loss of receptors could be
| Pretreatment (48 h) | Phosphoinositols (dpm) | % of control response | Significance (P) | |
|---|---|---|---|---|
| Basal | AII (100 nM) | |||
| None | 4,599 ± 188 | 16,316 ± 78 | 100.0 | Control |
| AII (10 nM) | 4,597 ± 168 | 13,703 ± 840 | 77.7 | <0.05 |
| Forskolin | 5,435 ± 121 | 8,721 ± 766 | 28.0 | <0.05 |
| (10 LM) | ||||
H295 cells were treated without or with AII (10 nM) or forskolin (10 uM) during the 48-h labeling period (in medium containing 10 uCi/ml [3H]inositol). Medium was removed, and cells were washed and incu- bated in medium containing 10 mM LiCl as described. Cells were then treated with or without 100 nm AII for 30 min, and the radioactive phosphoinositols were recovered and quantified as described. Results are the mean ± SEM of data from three wells and are from one of two similar experiments. Significant differences in response from control cells (100%) are indicated.
120
100
% of Control Cells
80
Z
60
AT,-R mRNA
40
A
A
4
1251-All Bound
20
0
0
12
24
36
48
Forskolin Treatment (h)
detected within 6 h of treatment with forskolin (Fig. 3), becoming maximal by 12 h, with no subsequent recovery. A rapid decline in mRNA levles was also observable within 0.5 h, becoming maximal by 6 h and showing some recovery to 50% below control values thereafter. Similar results were obtained for treatment with (Bu)2CAMP (not shown). The effects of All on levels of AT1-R mRNA were equally rapid (Fig. 4a), with almost maximal loss of message detected by 6 h, and thereafter almost fully returned to control levels at times beyond 12 h (Fig. 4a). The effects of All on levels of
a)
100
80
AT1-R mRNA (% control counts)
60
40
20
0
0
12
24
36
All Treatment (h)
b)
100
80
AT1-R mRNA (% control counts)
60
40
20
0
Control
-11
-10
-9
-8
-7
Log[AlI] (M)
AT1-R mRNA were also found to be dose dependent (Fig. 4b); there was a 50% drop in message after 4 h with as little as 10-11 M AII, suggesting that the response was physiologi- cally relevant.
The mechanisms of action of AII on reduction in AT1-R mRNA were studied in comparison to the effects of the calcium ionophore (A23187) and phorbol ester (TPA). Al- though these two agents acting alone could reduce the level of AT1-R mRNA, only in combination could they fully repro- duce the action of AII at 4 h of treatment (Fig. 5).
The effects of cycloheximide on changes in AT1-R mRNA after treatment (4 h) with forskolin and AII were also ex- amined (Fig. 6). The presence of cycloheximide (35 uM) was
120
100
AT1-R mRNA (% control counts)
80
60
40
20
0
Control
A23187
TPA
A23187/TPA
All
effective in opposing the observed decrease in message in response to AII, but not in response to forskolin.
Discussion
Although the development of specific antagonists to AT1- R and AT2-R and the cloning of the type 1 receptor in many species have allowed a greater knowledge of the distribution and possible function of AII receptor subclasses, little is known of the regulation of their expression. AII plays a pivotal role in adrenocortical cell production of aldosterone through the activation of AT1-R (15-17). One level at which the effects of AII could be regulated is through changes in the expression of AT1-R on the adrenocortical cells. Limited data are available from in vivo studies in rats suggesting that AII may up-regulate levels of AT1-R and its corresponding mRNA in the adrenal (11, 18, 19), but in rat kidney glomer- ular mesangial cells, both AII and (Bu)2CAMP reduce AT1A- R mRNA levels through apparently different mechanisms (20). A more recent study in human adrenocortical cells has also suggested that AII may slowly down-regulate AT1-R mRNA levels by 40% over 4 days (12). Thus, there may be both tissue- and species-related differences in the regulation
120
100
AT1-R mRNA (% control counts)
80
60
40
20
0
CX
-
+
-
+
-
+
Control
All
Forsk
of AT1-R mRNA levels. Furthermore, no information has been presented in these previous studies relating changes in mRNA levels with changes in AII binding and activation of phosphoinositidase-C.
In this study we examined the effects of activators of the protein kinase-A and the calcium/protein kinase-C pathways on AT1-R mRNA, binding, and activation of phosphoinosi- tidase-C in a human adrenocortical carcinoma cell line (NCI- H295 cells). We have previously established that these cells respond to AII stimulation with phosphoinositidase-C acti- vation and increased intracellular free Ca2+ concentration as well as increased production of aldosterone and expression of aldosterone synthase (13). Our results shown here reveal that activation of the adenylate cyclase/protein kinase-A pathway rapidly decreased levels of AT1-R mRNA, and this was paralleled by a loss of both AT1-R binding and phos- phoinositidase-C response. This loss of AII-binding sites in response to protein kinase-A activators is similar to that reported previously for bovine adrenocortical cells (21). However, although treatment of H295 cells with AII also rapidly decreased AT1-R mRNA levels in a dose-dependent manner and at physiological concentrations of AII, there was little corresponding effect on AT1-R binding or the phos- phoinositidase-C response. This difference in the action of AII and forskolin on receptor binding may relate to the deduced amino acid sequence in the C-terminus of the receptor protein, where there are multiple sites for protein kinase-C action, but not protein kinase-A (7, 22). Thus, the effects of agonists on AII receptor expression may be me-
diated by multiple signaling pathways acting at many levels in human tissues.
In many tissues, AII treatment can result in both increased intracellular Ca2+ concentration and increased production of diacylglycerol. This may lead to activation of protein kinase- C and/or activation of other calcium-dependent protein kinases. The mechanism of action of All on levels of AT1-R mRNA in H295 cells was fully reproduced by the action of phorbol ester in combination with calcium ionophore, sug- gesting that the action of All on AT1-R mRNA involved the activation of protein kinase-C, but may require activation of additional calcium-dependent protein kinases.
Control of mRNA levels in cells can be achieved by many processes, including altered rates of transcription or degra- dation. Such effects are often mediated through rapidly synthesized proteins, which have a short half-life. The effec- tiveness of cycloheximide in preventing the loss of AT1-R mRNA after treatment with AII suggests that control of AT1- R expression is at least partly achieved through a process that requires ongoing synthesis of such a protein. However, the finding that cycloheximide could largely reverse the effect of AII, but not forskolin, also underlines the different mech- anisms of action of All and forskolin in controlling functional AT1-R expression, a finding similar to that reported in rat kidney mesangial cells (20). Whether the putative protein factor acts to regulate transcription or, alternatively, promotes degradation of existing message remains unknown. It is interesting to note, however, that the 3’ cDNA sequences published for human AT1-R include six ATTTA sequences, corresponding to AUUUA destabilization mRNA sequences (7, 23).
In conclusion, our findings demonstrate that hormonal control of AT1-R expression occurs in response to both the protein kinase-A and protein kinase-C/calcium signaling pathways in the H295 human adrenocortical carcinoma cell line and suggest that the mechanisms controlling receptor expression are complex, operating on many levels. These findings contrast to that found previously for changes in AT1B-RmRNA in the rat adrenal, but are largely in agreement with the changes in AT1A-R mRNA observed in rat kidney mesangial cells (20). This confirms that differences exist between species in the mechanisms of regulation of AT1-R expression and suggests that the NCI-H295 cell provides a valuable model system to study the mechanisms operating in the human. Together with our previous observations (13, 14), our findings also confirm that the NCI-H295 cell line provides a much needed model system for the study of mechanisms underlying the control of aldosterone secretion in the human.
Acknowledgments
The authors would like to thank Dr. Mala Mahendroo and Jo Corbin for their assistance.
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