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Localization of ACTH receptor mRNA by in situ hybridizationin mouse adrenal glandYun Xia, Jarl E. S. Wikberg

Department of Pharmaceutical Biosciences, Division of Pharmacology, Box 591, Biomedical Centre, Uppsala University,S-751 24 Uppsala, Sweden

&misc:Received: 4 March 1996 / Accepted: 21 May 1996

&p.1:Abstract. Stimulation of steroidogenesis in the adrenalcortex is a major physiological action of adrenocortico-tropic hormone (ACTH). This action is presumed to bemediated by the ACTH receptor and is functionally con-nected with the hypothalamus-pituitary-adrenal axis. Togain information concerning the distribution of theACTH receptor in this axis, we examined mRNA for theACTH receptor in the adrenal gland, pituitary and hypo-thalamus of the mouse, by using in situ hybridization.The Y-1 mouse adrenal tumour cell line was also exam-ined. The specific hybridization signal for ACTH recep-tor mRNA was uniformly distributed in the Y-1 cells, al-though the level of expression was low. The adrenal cor-tex showed a strong signal for the ACTH receptor mR-NA in both the zona fasciculata and the zona glomeru-losa, sites that are involved in the mediation of the actionof ACTH on the synthesis and release of glucocorticoidsand aldosterone. In the zona reticularis of the cortex, asmall number of cells showed positive hybridization sig-nals. Moreover, a few scattered cells were positive in theadrenal medulla. In contrast, the hybridization results forboth the hypothalamus and the pituitary proved to benegative. These results indicate that the mouse ACTHreceptor is a peripheral receptor that is exclusively locat-ed in the adrenal gland.

&kwd:Key words: ACTH – ACTH receptor – Y-1 cell – Adre-nal cortex – Hypothalamus – Pituitary – Mouse (Y-1,CS7BL/6)

Introduction

It is generally believed that adrenocorticotropic hormone(ACTH) stimulates corticoadrenal steroidogenesis bybinding to its specific receptors in the adrenal cortex,thereby allowing the release of both glucocorticoid andmineralocorticoid hormones. Previous studies involvingthe use of 125I-labelled ACTH or derivatives of ACTH,

such as [125I]-Phe2,Nle4 ACTH1–38 or [125I]-Phe2,Nle4

ACTH1–24, have demonstrated the existence of high af-finity binding sites for ACTH in cells of both the zonafasciculata and the zona glomerulosa of the adrenal cor-tex (Lefkowitz et al. 1970; McIlhinney and Schulster1975; Buckley and Ramachandran 1981). Recent molec-ular cloning has identified five types of melanocortin re-ceptor cDNAs (Mountjoy et al. 1992; Chhajlani andWikberg 1992; Gantz et al. 1993a, b; Chhajlani et al.1993). One of these cDNAs appears to encode a humanACTH receptor (Mountjoy et al. 1992), now also termedthe melanocortin-2 (MC-2) receptor. The mRNA for theACTH receptor has been found in the adrenal cortex ofthe rhesus monkey by use of a hybridization probe de-rived from the human ACTH receptor. More recently,bovine and mouse ACTH receptor cDNAs have beenisolated (Raikhinstein et al. 1994; Kubo et al. 1995;Cammas et al. 1995), the former showing 81% and thelatter 89% amino acid sequence identity with the humanACTH receptor. Mouse ACTH receptor cDNA is func-tionally expressed in Hela cells, where the increase incAMP production can be induced by ACTH in a dose-dependent manner, but not by α-MSH (Cammas et al.1995). On the other hand, accumulated evidence indi-cates that ACTH exerts a broad range of physiologicalaction in vivo, especially in the hypothalamic-pituitary-adrenal (HPA) axis. In the present study, we have exam-ined the localization of ACTH receptor (MC-2R) mRNAin the mouse adrenal gland, pituitary and hypothalamus,by using in situ hybridization with a specific oligonucle-otide probe designed according to the mouse ACTH re-ceptor cDNA sequence, in order to survey the distribu-tion of ACTH receptor mRNA in the HPA axis.

Materials and methods

Cell and tissue preparation

Y-1 mouse adrenal tumour cells were grown in Dulbecco’s modi-fied Eagle medium with 10% fetal calf serum, 100 IU penicillinand 100µg streptomycin at 37oC in an atmosphere of 5% CO2 inCorrespondence to: J. Wikberg&/fn-block:

Cell Tissue Res (1996) 286:63–68

© Springer-Verlag 1996

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humidified air. Subconfluent cells were harvested and placed ontopoly-L-lysine-coated glass slides with the culture medium de-scribed above. Attached cells were rinsed twice with 0.01 M phos-phate-buffered saline (PBS; pH 7.4) and fixed in 4% paraformal-dehyde for 10 min at 4oC and then stored at 4oC in 70% ethanolfor use within 24 h. Adrenal glands, pituitary glands and brainswere taken from adult female CS7BL/6 mice and immediatelyfrozen on crushed dry ice. The tissues were sectioned on a cryo-stat microtome at a thickness of 14µm and the sections werethaw-mounted onto poly-L-lysine-coated glass slides and stored at–80oC until used.

In situ hybridization

A 48-mer oligonucleotide probe for the mouse ACTH receptorwas designed based on the published cDNA sequence (Kubo et al.1995). This probe (5′-TGC CGC TCC CTG TGC AGA ACA TCCAGA TAA TTG TTA GGG TGA TGA TGG-3′), which was com-plementary to nucleotides 440–487 of the mouse ACTH receptorcDNA, was confirmed not to share significant sequence homologywith any other melanocortin receptor cDNA cloned to date (GEN-BANK/EMBL) by using computer-assisted sequence analysiswith MacMolly Tetra software. The probe was also predicted tohave satisfactory hybridization properties by this software. In situhybridization was carried out as previously described (Xia et al.1995), with the modification that the washing temperature was ad-justed to 53oC according to the calculated melting temperature ofthe probe. The synthesized oligonucleotide was labelled at the 3′

end with [α-35S]dATP (Amersham) by using terminal deoxynucle-otidyl transferase (Promega) and subsequently purified on aNENSORB 20 nucleic acid purification cartridge (NEN/DuPont).The sections were covered with hybridization solution, viz., 50%formamide, 4× SSC (1× SSC=150 mM NaCl, 15 mM sodium ci-trate, pH 7.0), 1× Denhardt solution (0.02% bovine serum albu-min, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 1% sarcosyl,0.02 M PBS (pH 7.0), 10% dextran sulphate, 300µg/ml yeasttRNA, 500 µg/ml denatured salmon testis DNA and 60 mM di-thiothreitol, containing 107 cpm/ml probe, and hybridized at 42oCovernight in a humidified chamber. The specificity of the hybrid-ization was controlled by treating parallel slides with a solutioncontaining a 100-fold excess of the unlabelled probe. After beingwashed 4 times (15 min each) at 53oC in 1×SSC, dehydration inethanol and air-drying, the slides were dipped in Kodak NTB2emulsion diluted 1:1 in water, exposed for 6 weeks at 4oC and de-veloped in Kodak D19 solution. The developed slides were coun-terstained with cresyl violet and examined using a Leitz DMR mi-croscope equipped with both bright- and dark-field condensers.The level of the mRNA signal was analysed by a computer-assist-ed image analysis system with NIH Image 1.54 software.

Results

Since the mouse ACTH receptor cDNA was isolatedfrom the Y-1 cell line, it was of interest to examine thesecells, not only for their level of expression of the ACTH

Fig. 1A, B. Expression of ACTH receptor mRNA in Y-1mouse adrenal tumour cells under bright- and dark-field,respectively. The hybridization signal is uniformly dis-tributed over the cells. Bar: 50 µm&/fig.c:

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Fig. 2A–F. Localization of ACTH receptor mRNA by in situ hy-bridization in the mouse adrenal gland. A, B Note the specific la-belling forming a ring covering the adrenal cortex (A dark-field, Bbright-field) at low magnification. C–F Detailed labelling of theadrenal cortex as seen at higher magnification. C, Adrenal cortex;M, adrenal medulla; G, zona glomerulosa; F, zona fasciculata; R,zona reticularis. Bars: 800µm in A, B; 200µm in C, D; 50 µm inE, F&/fig.c:

receptor mRNA, but also to assess the functionality ofthe oligonucleotide probe used. Specific ACTH receptormRNA hybridization signal was uniformly distributedover the Y-1 cells (Fig. 1A, B). However, the signal wasnot strong, indicating that the expression of the ACTHreceptor mRNA was low. In contrast to the results for theY-1 cells, the cortical region of the adrenal gland showedextremely high labelling for ACTH receptor mRNA.Abundant labelling consistently formed a “silver ring”that covered the adrenal cortex and that was easily ob-servable with a dark-field condenser (Fig. 2A, B). Athigher magnification (Fig. 2C–F), the hybridization sig-nal was localized to the zona glomerulosa and zona fas-ciculata of the cortex, the labelling being strong and uni-formly distributed over the cells. The zona reticularis,the thin innermost zone of the cortex, showed much lesslabelling that was restricted to small groups of cells(Fig. 3A). Interestingly, labelling was found in a few in-dividual scattered cells in the adrenal medulla (Fig. 3B).The capsula lying at the outermost zone of the cortexshowed no labelling. Similar negative results were ob-

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tained in the hypothalamus of the brain, including theperiventricular nucleus, the arcuate nucleus and theventromedial hypothalamus (Fig. 4A-D), and the pituita-ry gland, including the anterior, intermediate and poste-rior lobe (not shown).

Discussion

In situ hybridization in the present study has demonstrat-ed that mRNA for the ACTH receptor is highly ex-pressed in the adrenal cortex, with the most prominentexpression being confined to the zona glomerulosa andzona fasciculata. These results are consistent with earlierradioligand-binding studies (Lefkowitz et al. 1970; McIlhinney and Schulster 1975; Buckley and Ramachand-ran 1981) and with a recent study of the adrenal gland ofthe rhesus monkey (Mountjoy et al. 1992). The cells ofthe zona glomerulosa are involved in the secretion of al-dosterone, whereas the cells of the zona fasciculata areinvolved in the secretion of glucocorticoids. The cellularexpression of ACTH receptor mRNA in these regionsthus strongly supports the notion that the very sameACTH receptor is responsible for mediating the actions

of ACTH on the synthesis and secretion of both gluco-corticoid and mineralocorticoid hormones. In addition,some clustered cells in the zona reticularis of the cortexand a few scattered cells in the adrenal medulla appearto express mRNA for the ACTH receptor. This findingsuggests that these cells also have the capability of syn-thesising and secreting glucocorticoid hormones. Alter-natively, these cells could participate in actions involv-ing the secretion of sex steroids or catecholamines. Fur-ther studies are required to examine these possibilities.An interesting question answered in this study is wheth-er the ACTH receptor exists in the pituitary and/or thehypothalamus. Based on the fact that ACTH is an impor-tant mediator that is functionally connected with theHPA axis, it is possible that the ACTH receptor is alsolocated outside the adrenal gland and that it has a role inregulating ACTH secretion. Moreover, a previous radio-ligand-binding study has provided evidence that ACTH-binding sites are detectable in regions that include thehypothalamus in the rat brain (Hnatowich et al. 1989).However, we have failed to detect mRNA for the ACTHreceptor in the pituitary and the hypothalamus, suggest-ing that none of these areas express the ACTH receptor(MC-2R). These results are in agreement with the obser-

Fig. 3. Expression of ACTH receptor mRNA in the zonareticularis of the adrenal cortex (A) and medulla (B). Notethat a few cells are labelled (arrows). F, Zona fasciculata;R, zona reticularis; M, adrenal medulla. Bar: 50 µm&/fig.c:

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Fig. 4A–D. In situ hybridization localization of ACTH receptormRNA in the mouse hypothalamus. Note that no specific signalcould be identified in the anterior hypothalamus (A, B), includingthe paraventricular nucleus (PVN), medial preoptic area (MPA)and septohypothalamic nucleus (SHy), or in the tuberal hypothala-

mus (C, D), including the ventromedial hypothalamus (VMH),dorsomedial hypothalamic nucleus (DM) and arcuate nucleus ofthe hypothalamus (ARH). A, C Dark-fields. B, D Correspondingbright-fields to those shown in A, C, respectively. AC, Anteriorcommissure. Bar: 400µm

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vation of the localization of the ACTH receptor in therhesus monkey (Mountjoy et al. 1992) but are in conflictwith the radioligand-binding study of Hnatowich et al.(1989). The reason for this discrepancy could be the lim-itation of the receptor-binding assay, which may not beable to distinguish ACTH receptors from other melano-cortin receptors, such as the MC-3 and MC-4 receptors.Both the MC-3 and MC-4 receptor have recently beenreported to be present in the hypothalamus and to be ca-pable of binding ACTH with high affinity (Gantz et al.1993a, b; Roselli-Rehfuss et al. 1993; Mountjoy et al.1994; Schiöth et al. 1995). It is therefore possible thatthe binding sites detected by [125I][Phe2, Nle4]ACTH1–24may not be the ACTH receptor, but instead some of theother melanocortin receptors, such as the MC-3 andMC-4 receptor.

From the present study the following conclusions canbe drawn. Firstly, the ACTH receptor seems to be a pre-dominantly peripheral receptor expressed in the adrenalgland. Secondly, the ACTH receptor may not be capableof mediating central feedback regulation, as the receptorappears not to be present in the hypothalamus and the pi-tuitary. However, a negative feedback effect of ACTH oncorticotropin-releasing factor has been reported (Mottaet al. 1965; Suda et al. 1987). We therefore suggest thatthis feedback regulation of ACTH is mediated by eitherthe MC-3 and/or the MC-4 receptor, both of which arepresent in the hypothalamus. Thirdly, the finding that afew cells containing ACTH mRNA exist in the zona reti-cularis and medulla of the adrenal gland indicates thatACTH has a regulatory function in these cells. Finally,the high expression of ACTH receptor mRNA in themouse adrenal cortex suggests that mice represent agood model for studying the role of the ACTH receptorin the synthesis and secretion of corticosteroid hormonesduring various physiological and pathological condi-tions.

&p.2:Acknowledgements.This study was supported by a grant from theSwedish MRC (No. 04X-05957), and the Swedish Society forMedical Research.

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