Rapid Paper ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines
Kathleen G. Mountjoy ª, Ian M. Bird b, William E. Rainey *,b and Roger D. Cone ª
” Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon, USA;
b The Departments of Obstetrics & Gynecology, Biochemistry and The Cecil H. & Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas, USA
(Received 28 October 1993; accepted 18 November 1993)
Key words: Corticotropin; Corticotropin receptor; Adrenocortical cell line
Summary
Corticotropin (ACTH) binds to specific receptors in the adrenal cortex and thereby regulates glucocorticoid and mineralocorticoid production. The number of ACTH binding sites on adrenocortical cells is increased by exposure of cells to activators of the cAMP pathway. The mechanism responsible for the increase in ACTH binding sites is not known. We therefore studied the levels of ACTH-R mRNA in mouse Y-1 and human NCI-H295 (H295) adrenocortical carcinoma cell lines. ACTH induced an increase in mouse ACTH-R mRNA in Y-1 cells that was time and dose dependent, increasing 6-fold over basal levels following exposure to 10-% M ACTH for 19-24 h. The amount of human ACTH-R mRNA in H295 cells increased 2-4-fold following a 24 h exposure to 10-8 M ACTH, 1 mM dbcAMP, or 10-5 M Forskolin. Treatment of H295 cells with angiotensin II (A-II) was found to dramatically increase the level of ACTH-R mRNA. These data indicate that regulation of ACTH-R mRNA levels is at least one mechanism by which ACTH and A-II elevate the number of ACTH binding sites in the adrenocortical cell.
Introduction
Corticotropin (ACTH) acutely regulates glucocorti- coid and mineralcorticoid production in mammals by binding to specific receptors on the adrenal cortex and activating adenylyl cyclase (Grahame-Smith et al., 1967; Buckley et al., 1981; Mertz et al., 1991). Over a longer period of time, ACTH increases the glucocorticoid output of adrenal fasciculata cells by increasing expres- sion of several steroid metabolising enzymes (Simpson et al., 1988). Additionally, ACTH increases adrenal cortex size and is crucial for the normal development of this tissue (Allen et al., 1974; Ballard et al., 1980).
The expression of ACTH binding sites on human, ovine and bovine adrenocortical cells has been re- ported to be up-regulated by exposure of cells to ACTH (Penhoat et al., 1989a,b; Rainey et al., 1989, 1991) through the protein kinase A (PKA) pathway. A number of other hormones and growth factors have also been reported to control the number of ACTH
binding sites on the the adrenal cortex. Prostaglandin E2, for example, up-regulates ACTH binding on adrenal cells (Rainey et al., 1991) probably also through the PKA pathway. Insulin-like growth factor I (IGF-I) has been demonstrated to increase ACTH binding to cultured bovine adrenocortical cells (Penhoat et al., 1989a,b) and this effect is synergistic with ACTH in- duced up-regulation of ACTH receptors. In contrast, transforming growth factor ß (TGFB), which is a po- tent inhibitor of adrenocortical cell differentiated func- tion, down-regulates ACTH binding in bovine adreno- cortical cells and blocks the ability of ACTH to up-reg- ulate ACTH binding (Rainey et al., 1989). The ACTH- R gene has recently been cloned (Cone et al., 1992; Mountjoy et al., 1992), which has made feasible this study of the effects of ACTH on ACTH-R mRNA expression.
Materials and methods
Cell culture Mouse Y-1 and human NCI-H295 adrenal tumor cells were obtained from the American Type Culture Collection (Rockville, MD). Mouse Y-1
* Corresponding author. Tel .: 214-688-3266; Fax: 214-688-8066.
cells were maintained in Dulbecco’s modified Eagles F10 medium (DME-F10) containing 15% heat inacti- vated horse serum (HS), 2.5% heat inactivated fetal calf serum (FCS) and antibiotics. Human NCI-H295 cells were maintained in an equal mixture (V/V) of DME and F12 media containing 1% ITS plus (Col- laborative Research, Bedford MA; 1 mg/ml insulin, 1 ng/ml selenium, 1 mg/ml transferrin, 1 mg/ml linoleic acid, 1.25 mg/ ml BSA), 2% Ultroser SF (Sep- racor, Marlborough MA) and antibiotics (defined medium). Mouse Y-1 cells were grown in 100 mm culture plates until they reached approximately 70% confluency. Medium was then changed to F10 with 0.5% HS for 24 h. Cells were then treated with ACTH (Bachem, Torrance CA) or dibutyryl cAMP (dbcAMP) in medium containing 0.1% BSA. Human NCI-H295 cells were subcultured and, after 48 h, rinsed and placed in fresh defined medium and treated with ACTH, forskolin, or dbcAMP.
Probes for Northern analysis A human ACTH-R 400 bp DNA probe was originally obtained by poly- merase chain reaction (PCR) with human melanoma cDNA as a template and degenerate oligonucleotides to conserved regions in transmembrane domains 3 and 6 of G-protein coupled receptors as previously de- scribed (Mountjoy et al., 1992). A mouse ACTH-R 200 bp DNA probe was obtained by PCR with mouse adrenal cDNA as a template and degenerate oligonu- cleotides to conserved regions in the second and third intracellular loops of the human ACTH and MSH receptors. The second intracellular loop oligonu- cleotide was:
5’-GAGTCGAC(ACT)TT(CT)CCUGC(ACGT)(C- T)TI(AC)G (ACGT)TA(CT)CA(CT)-3’, and the third intracellular loop oligonucleotide was:
5’-CAGAATTCAT(ACGT)GT(ACGT)A(A - G)IGT(ACGT)A(CT)IGC(ACGT)CC(CT)-3’. Brackets indicate variable nucleotide positions and I indicates an inosine nucleotide. The PCR conditions were 94℃, 1 min, 45°℃, 2 min, 72℃, 2 min for 40 cycles. Probes were verified by DNA sequencing.
Isolation and analysis of cellular RNA Monolayers of Y-1 cells were solubilized in 4 M guanidine thio- cyanate. The RNA was then extracted by the method of Chirgwin (Chirgwin et al., 1979). RNA from human H295 cells was recovered by phenolic extraction in the presence of guanidine thiocyanate, as previously de- scribed (Bird et al., 1992). RNA (20 µg) was size separated by electrophoresis on denaturing 1.2% agarose formaldehyde gels. The integrity of the riboso- mal RNA species were examined under UV light to ensure consistency between lanes. RNA was then transferred to nylon filters, hybridized in 50% for- mamide, 1 M NaCl, 50 mM Tris (pH 7.5), sodium pyrophosphate (0.1%), SDS (0.2%), salmon sperm DNA (100 mg/ml), and 10 × Denhardt’s with either
the mouse ACTH-R 32 P-labelled DNA probe or the human ACTH-R 32 P-labelled DNA probe at 42℃ for 18 h. The filters were washed under stringent condi- tions (0.1 × SSC, 65°℃), exposed to Kodak XAR-5 film at - 80°℃ and analyzed by scanning densitometry.
Results
ACTH up-regulates ACTH-R mRNA expression in mouse Y-1 cells ACTH-R mRNA was increased a maximum of 6-fold over basal following exposure of mouse Y-1 cells to 10-& M ACTH for 19-27 h in the absence of serum (Fig. 1). A single transcript of ap- proximately 2 kb was detected in the mouse. This increase in ACTH-R mRNA was first detected approx-
2
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0
kb
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1.4-
0 .5 1 1.5 2 4 7 19 24 27 27 Hrs
15% HS
0.1% BSA + 10-8M ACTH
0.1% BSA
imately 4-7 h after ACTH treatment, and was stable even over a period of up to three days. ACTH induced ACTH-R mRNA expression was dose-dependent and was detectable with 10-9 M ACTH (not shown). Inter- estingly, in some experiments a small induction of ACTH-R mRNA levels was seen with the withdrawal of serum alone, however induction specific to ACTH can be seen by comparing ACTH mRNA levels in cells grown in the absence of serum for 27 h, with or without 10-M ACTH treatment (Fig. 1, last 2 lanes).
8
Arbitrary units
6
4
2
0
kb
4.4 -
2.4 -
1.4 -
1
control
3 6 12 24
ImM dbcAMP
10-8 M ACTH
Hours
(10-5 M Forskolin)
3
Arbitrary units
2
1
0
kb
— 4.4
-2.4
- 1.4
0 -11 -10 -9 -8
[Angiotensin II] (logM)
ACTH, Forskolin, dbcAMP, and AII up-regulate ACTH-R mRNA in human H295 cells Exposure of H295 cells for 24 h to ACTH (10-8 M) increased ACTH-R mRNA expression 1.2-fold over basal (Fig. 2). Because the response to ACTH is low in the H295 cell line (Rainey et al., 1993), we also examined the ability of other activators of the cAMP signal transduc- tion pathway to elevate ACTH-R mRNA. ACTH-R mRNA was induced 3-fold over basal in H295 cells following exposure to 1 mM dbcAMP for 24 h.
Forskolin (10-5 M) also induced both ACTH-R mRNA transcripts in H295 cells. This increase was first detected after 6 h and peaked with a 4-fold induction over basal at 24 h (Fig. 2). Two transcripts were seen at approximately 2 and 4 kb, consistent with previous reports for human adrenal (Cone and Mountjoy, 1992). Both transcripts appear equally induced by ACTH suggesting that these transcripts are under the tran- scriptional control of a common regulatory unit.
In contrast to the mouse Y1 cell line, H295 cells respond to A-II with increased mineralocorticoid pro- duction, and thus serve as a model for adrenal
glomerulosa cells (Bird et al., 1993) Interestingly, A-II was found to induce a significant elevation in ACTH-R mRNA levels in the H295 cells (Fig. 3). The induction was dose-dependent, detectable following a 24 h treat- ment with 10-9 M A-II, and peaking with 10~8 M A-II treatment.
Discussion
We have shown that ACTH up-regulates ACTH-R mRNA levels through activation of the cAMP pathway in both mouse Y-1 and human H295 adrenocortical cells. Increased ACTH binding and responsiveness have previously been reported following chronic exposure of human (Rainey et al., 1991) and bovine (Penhoat et al., 1989a,b) adrenal cells to ACTH. We therefore expect that the increase in ACTH-R mRNA we have observed is reflected in a corresponding increase in ACTH-R protein. However, confirmation of this, and the time course of protein regulation, must await the availability of specific ACTH-R antibodies.
The studies presented here do not address the mechanism by which ACTH-R mRNA levels are ele- vated by cAMP. If the ACTH-R gene is regulated as are many of the steroidogenic enzyme genes (for re- view, see (Simpson and Waterman, 1992)), then it is likely that much of the regulation is transcriptional. Sequencing of 781 bp upstream of the start of transla- tion of the human ACTH-R has not yet identified a “classical” CRE consensus sequence (KGM & RDC, Genbank/ EMBL Accession X65635), however this is also consistent with the steroidogenic enzyme genes, which are regulated by cAMP but do not have func- tional CRE sequences (Simpson and Waterman 1992).
ACTH is known to transiently stimulate mineralo- corticoid production, although the physiological rele- vance of this is unclear. Consequently, induction of ACTH-R mRNA levels by A-II may serve to further enhance mineralocorticoid output. Since A-II acts pri- marily via Ca2+ and diacylglycerol, it is likely that the ACTH-R gene is regulated by multiple signal transduc- tion pathways, which might explain the ability of so many different agents to regulate the number of ACTH binding sites (Penhoat et al., 1989a,b; Rainey et al., 1989, 1991).
Agonist induced up-regulation of a receptor by its ligand represents a novel regulatory phenomenon; pre- sumably the increased responsiveness to ligand that
results plays a physiological role in regulating the ulti- mate output of glucocorticoids, and perhaps mineralo- corticoids produced by the adrenal cortex. This finding also implies the existence of downregulatory mecha- nism(s) that would be needed to attenuate autologous induction in this system.
Acknowledgements
This work was supported by grants to W.E.R. (American Heart Association Texas Affiliate 93R-082), and R.D.C. (NIH PO1ADK44239).
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