185 Eph receptors and zonation in the rat adrenal cortex

Caroline H Brennan, Alexandra Chittka, Stewart Barker and Gavin P Vinson School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, UK (Correspondence should be addressed to G P Vinson; Email: [email protected])

Abstract

Although the zonation of the adrenal cortex has a clear zona glomerulosa. Its transcription closely reflected expected functional role, the mechanisms that maintain it remain changes in the glomerulosa phenotype, thus it was increased largely conjectural. The concept that an outer proliferative after a low-sodium diet, but decreased by pretreatment with layer gives rise to cells that migrate inwards, adopting the angiotensin-converting enzyme inhibitor, captopril. It sequentially the zona glomerulosa, fasciculata and reticularis was also decreased by ACTH treatment, but unaffected by phenotypes, has yet to be explained mechanistically. In other betamethasone. mRNA coding for A1, the major tissues, Eph (EphR)/ephrin signalling provides a for the EphA receptors, was also detected in the rat mechanism for cellular orientation and migration patterns. adrenal, though changes evoked by the various pretreatments Real-time PCR and other methods were used to determine did not clearly reflect the expected changes in zonal function. the possible role of Eph/ephrin systems in the rat adrenal. Because the maintenance of cellular zonation requires clear mRNA coding for several members of the EphR family was positional signals within the adrenal cortex, these data support detected, but out of these, EphA2 provided the closest parallel a role for Eph forward and reverse signalling in the to zonal organisation. In situ hybridisation showed that EphA2 maintenance of adrenocortical zonation. mRNA and EphA protein were predominantly located in the Journal of Endocrinology (2008) 198, 185–191

Introduction sequentially adopts the phenotypes of glomerulosa, fasciculata and reticularis cells during its migration through the gland. Aspects of adrenocortical zonation remain enigmatic. We have argued that an implication of this mechanism is that Functionally, the accepted view is that the glomerulosa is individual adrenocortical cells have a polarity, each with apical the source of aldosterone secretion and the fasciculata and and basal borders facing the capsule and medulla respectively reticularis are the source of glucocorticoids (Deane 1962, (Vinson & Ho 1998, Vinson 2003). Ogishima et al. 1992, Vinson et al. 1992, Ennen et al. 2005) This poses the question: what are the locally generated and (depending on species) androgens as well (Deane 1962, signals that determine phenotypic changes at specific sites in Parker et al. 1983, Vinson et al. 1992, Rehman & Carr 2004). the gland? Is it attributable to a morphogenic landscape, in Within this system, aldosterone secretion is under the which non-cortical cells, such as neural or vascular cells, regulation of angiotensin (and other agents) and adrenocorti- release local organisers that vary in nature at different latitudes cotrophin (ACTH) supports fasciculata and reticularis (Ho & Vinson 1997, Vinson & Ho 1998)? Alternatively, does functions. Though this simple model probably needs the signalling between adjacent cortical cells, linked to their adjustment and reappraisal (Vinson 2003, 2004), it never- polarity, provide sufficient information to promote specific theless seems evident that the zonal arrangement is important cellular phenotypes at specific loci? in the maintenance of adrenocortical function, so it is That the polarity of the adrenocortical cell cords, signalling important to determine how it arises. centripetally via cell–cell contact, might in combination give The cell-migration theory, first proposed by Gottschau sufficient information to establish zonation has not been (Deane 1962, Gottschau 1883), holds that the outermost part considered, though related processes have been described in of the cortex is the major site of adrenal cell proliferation, and other tissues, in which the Eph receptor (EphR)/ephrin the inner zones are the site of cell death. Though there are signalling system has been shown to have a special significance. conflicting views (Wolkersdorfer & Bornstein 1998, Mitani In particular, in the small intestine of the mouse, progeny of the et al. 2003), it does seem likely that adrenocortical cells are stem cells that border the intervillus pockets migrate in precise propagated centripetally, in cords, through the cortex patterns. Wnt signalling results in b-catenin stabilisation and (Wright & Voncina 1977, Bertholet 1980, Zajicek et al. interaction with T-cell factor (TCF) transcription factors, 1986, McNicol & Duffy 1987, Stachowiak et al. 1990, Morley leading in turn to inverse regulation of EphB2/B3 receptors et al. 1996). Consequently, each adrenocortical cell and their ligand (Batlle et al.2002). The receptors

Journal of Endocrinology (2008) 198, 185–191 DOI: 10.1677/JOE-08-0084 0022–0795/08/0198–185 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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restrict cell intermingling and distribute cell populations to briefly at Queen Mary, University of London under standard discrete locations within the intestinal epithelium. Similar conditions of light and temperature, in accordance with expression gradients of EphRs and ligands may determine tissue appropriate guidelines for animal care. As required, animals patterning in other systems, for example in the subventricular received treatments as follows: ACTH-treated animals were zones ofthe brain in which EphA/ephrin signalling regulates cell injected 5 days subcutaneously with 100 mgDepot migration and the balance between proliferation and differen- Synacthen (Ciba-Geigy, UK) and betamethasone (Betnesol, tiation, or in the topographic maps of neuronal connections Glaxo-Wellcome, UK) was provided in drinking water, (Conover et al.2000, Wilkinson 2001, Kullander & Klein 2002, 0.2 g/100 ml, for 7 days. Captopril was also supplied in Pasquale 2005). drinking water (0.5 mg/ml) for 2 weeks. In all of these cases, As Wnt, b-catenin and TCF-like transcription factors controls were untreated. All of these animals were interact with steroidogenic factor (SF-1; Gummow et al. maintained on standard laboratory diets. For dietary sodium 2003, Kennell et al.2003) and have crucial roles in adrenal studies, control rats were maintained on a diet of wholemeal (and gonadal) development (Luo et al.1995, Parker et al.1995, flour (Sainsburys, Ltd, London, UK) supplemented with 1% Heikkila et al.2002, Keegan & Hammer 2002, Jeays-Ward et al. CaCO3 and 1% NaCl, with access to distilled water for 2003, Else & Hammer 2005), it is appropriate to examine the 3 weeks. The low-sodium diet omitted the 1% NaCl. possibility of EphR/ephrin signalling in the rat adrenal cortex. Animals were killed by stunning and cervical dislocation, To demonstrate the possibility of specific EphR/ephrin and adrenals were snap frozen and stored in liquid nitrogen, involvement, the tissues were taken from control animals, and or fixed in 4% (w/v) paraformaldehyde in PBS and stored at from animals subjected to treatments known to have specific 4 8Cpriortoin situ hybridisation. actions on adrenal cell differentiation and phenotype, and thus zonal expression. These were low-sodium diet or ACTH Preparation of RNA and real-time PCR treatment, which enhance glomerulosa and fasciculata expression respectively, and treatment with the angiotensin Tissues were homogenised in Trizol (Invitrogen) using an converting enzyme (ACE) inhibitor captopril or betametha- Ultra-Turrax T25 polytron homogeniser and RNA extracted sone, which reduce the circulating levels of the major stimulants according to the manufacturers’ instructions. RNAs were of the glomerulosa and fasciculata, namely angiotensin II and treated with 5 U/ml RNase-free DNase for 20 min at room ACTH (e.g. Vinson et al.1992, Raza et al.2005). temperature to destroy genomic DNA. Complete cDNA was synthesised from 1 mg of each RNA using random hexamers (500 nM, Promega), RNAsin (40U, Promega) and Super- Materials and Methods script III Reverse Transcriptase (Invitrogen) for 60 min at 50 8C according to the manufacturers’ instructions. For Animals EphR, primers used for PCR were as published previously Adult male Wistar rats 12–14 weeks old weighing 180–220 g (Biervert et al. 2001) and are given below (Table 1). Primers were obtained from commercial suppliers, and maintained used for ephrin PCRs are given in Table 1. Standard curves

Table 1 Real-time PCR: primers

Forward primer Reverse primer

Gene name EphrinA1 CCA ACA TTA CGA GGA CGA CTCT GGG CTC GCA TGT CAC ATA CTC EphrinA2 AGT CTA CTG GAA CCG CAG CAA TAG CCG CCG CCA TCA C EphrinA3 CCA TGC CGG CCA AGA A CCT TCA TCC TCA GAC ACT TCC AA EphrinA4 CAG CGG GAA CGG TGT GA GCT GGA ATT GCA CGC TAC CT EphrinA5 CGC TAT GTC CTG TAC ATG GTG AA TTC CCA TCT CTT GAA CCC TTT G EphrinB1 CGT AAC GCC TGA GCA GTT GA AGC CTG TGT GGC TGT CTT GAC EphrinB2 CCT ACA GAG CAC ATG GAA ACG A GCC AGA GAG ATC CCA TCA ATT C EphrinB3 TTC TGC GAG TGG GAC AAA GTC GGT CTC TCT CCA TGG GCA TTT EphA2 CCC GAG TGT CCA TTC GGC TAC TCA CTT GGT CTT TGA GTC CCA G EphA3 AGT CTG AAG ATC ATC ACA AGC CAC ATC CTT CCA GTA CTT TAC AC EphA4 AGT TCC AGA CCG AAC ACA GCC TTG GCC ATG CAT CTG CTG CAT CTG EphA5 CGC GTC AAG CAG GGT ATC TAC T ACC AGT ACT TTG CCA AGG GTT G EphA6 GAC ATC CTC GTA ATG CCA GAA T CGT CTA ATA TCG TCG ATG CTC A EphA7 CCA TAA GCC CTC TTC TGG ACC A ACA CTT GGA TGC CGG TTC CGT EphB1 ACC ATC ACC GCT GTG CCT TCC TTC TCA TGC CAT TAC CGA CGG TGA EphB2 CAC TAC TGG ACC GCA CGA TAC TCT ACC GAC TGG ATC TGG TCC A EphB3 CAG TGC CCC ATC TGG CAT GT CTT AGC AGA TCT TCT GCA GTC A EpHB6 GGA CAG GCC TTC CCA GGC TCT TGG CAG GTC TTC CAG GCT GA GAPDH CCC TCA AGA TTG TCA GCA ATG C GTC CTC AGT GTA GCC CAG GAT

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Downloaded from Bioscientifica.com at 09/29/2021 11:52:35AM via free access Eph receptors and zonation in rat adrenal cortex . C H BRENNAN and others 187 were used to establish empirically the appropriate PCR animals on each of at least 3 separate days with reverse conditions for each of the genes reported before comparative transcriptase-free samples for each RNA acting as negative experiments were carried out. Total RNA concentrations controls. Primer sequences are shown in Table 1. were quantified using an Eppendorf UV Biophotometer (Merck) and 1 mg was used per cDNA synthesis. For In situ hybridisation (ISH) GAPDH, cDNA was quantified similarly and 100 ng cDNA was used per reaction. Initial 40 cycle 20 ml PCRs were Sixteen micrometre sections of paraformaldehyde-fixed performed for each primer pair using cDNA from rat adrenal paraffin wax-embedded glands were cut using a Leica glands or brain with 0.5 mM of each primer prior to RM2145 (Leica Microsystems (UK), Milton Keynes,UK) performing quantitative real-time PCR. For those genes and in situ hybridisation performed as described elsewhere found to be expressed in the adrenal gland, parallel 25 ml real- (Knoll et al. 2001, Polvani et al. 2003). Digoxigenin-labelled time PCRs were set up each containing 1 ml (25 ng) cDNA RNA in situ probes were generated from 1 kb PCR and 300 ng of each primer. A 40 cycle PCR was performed at fragments of rat EphA2 and EphA3 cloned in pGEM-T 60 8C on a Stratagene MX3000P QPCR system (Stratagene, easy (Promega) vector. The primers used to generate the Cedar Creek, TX, USA) followed by a thermal dissociation cloned fragments were EphA2: forward 50-TGG GAC CTG- step to allow analysis of the product for purity. DNA synthesis ATG CAA AAC AT-30, reverse 50-CCA CCT TCT CGT- was monitored using SYBR green (Stratagene). Preliminary AGC CTT CTT-30 and EphA3: forward 50-TTG TCA- experiments confirmed that GAPDH mRNA itself was CCT CTC CAT CCT CAT-30, reverse 50-GAT AAC ATT- unaffected by the treatments and normalisation of candidate TCT TGG TGC GG-30. Signals were detected using alkaline gene expression against GAPDH was used to permit phosphatase conjugated anti-digoxigenin (Roche) and BM comparison between cDNAs. Each measurement was purple substrate (Roche). Sense probes were used as a performed in triplicate on the tissue from four different negative control.

Figure 1 PCR analysis of EphA and EphB receptor transcription in rat adrenal gland (Ad) and brain (Br). Reverse transcription-free samples (KRT) acted as negative controls. Size markers are 100 bp ladder (Smart ladder, Eurogentech). When using brain cDNA as the template, 200–300 bp products were detected for each receptor. EphA2, 3, 4 and 7, and EphB2, 3 and 6 are expressed in the adrenal gland. www.endocrinology-journals.org Journal of Endocrinology (2008) 198, 185–191

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Localisation of EphA receptor protein enhanced 50% by a low-sodium diet, but reduced by captopril to 75% of control values. The transcription of other subtypes Ligand binding domain alkaline fusion proteins (LAP) were was variably affected or unaffected (not shown). Based on generated as described previously ((Flanagan & Leder 1990, these preliminary data, subsequent experiments focussed on Brennan et al.1997) and used according to published procedure EphA2 expression. Figure 2 shows that EphA2 receptor (Brennan et al.1997, Brennan & Fabes 2003). LAP binding was transcription is localised in the glomerulosa, as is also the performed on 16 mmcryosectionsoffixedtissue. primary site for ephrin A ligand binding, which is an indirect indication of the presence of EphA receptor. The transcrip- tion of mRNA coding for the A3 subtype also mostly occurs Results in the glomerulosa (Fig. 3), although in this case unaffected by a low-sodium diet or captopril (data not shown). Eph receptors Finally, further analysis of the regulation of EphA2R transcription is consistent with its glomerulosa localisation, Evidence for transcription of mRNA coding for several since ACTH pretreatment, which may be expected to EphR subtypes in the rat adrenals is shown in Fig. 1. EphA2, enhance the fasciculata at the expense of glomerulosa A3, A4 and A7, and EphB3 and B6 are all expressed in the rat function, diminishes EphA2R mRNA production, although adrenal gland. In a preliminary set of experiments, changes in betamethasone treatment has no effect (Fig. 4). EphR expression levels in response to prior dietary sodium restriction or captopril treatment were determined. Of those EphR expressed within the adrenal, only EphA2 gene transcription showed responses that are clearly associated Ephrin ligands are expressed in adrenal tissue with ephrinA1, with zona glomerulosa phenotype, in that transcription was the predicted cognate ligand for EphA2, showing the highest level of expression (Fig. 5A). Real-time PCR also identified in adrenal tissue, and Fig. 5B shows the changes evoked by the different treatments.

Discussion

Ten EphA and six EphB receptors have been identified in vertebrates, classified according to their binding preferences for either the six glycosylphosphatidylinositol-anchor-linked ephrin A ligands or the three transmembrane ephrin B ligands. The significance of these interactions for cellular organisation and differentiation is that signalling between adjacent cells can occur in two directions, ‘forward’via the EphR and ‘reverse’via the ephrin ligand, but EphR/ephrin signalling can evoke repulsion as well as adhesion and attraction, thus governing cell migration, and leading to the possibility of cell segregation and the establishment of discrete populations of similar cells (for reviews, see Kullander & Klein 2002, Pasquale 2005). Clearly, such signalling could have the capacity to confer on adrenocortical cells, and on the cords they form, the polarity

Figure 2 (A and B) In situ hybridisation to EphA2 mRNA in the rat adrenal gland. (A and C) Antisense probes showing strong expression (dark stain) in the zona glomerulosa (arrow). The medulla (M) shows no reaction. (B and D) Sense probe. (E and F) Localisation of EphAR protein using alkaline phosphatase (AP) tagged ligand binding domain fusion proteins. Sections of gland were incubated in (E) AP-tagged soluble ephrinA5 or (F) soluble alkaline phosphatase as a negative Figure 3 In situ hybridisation to EphA3 mRNA. (A) Antisense probe control. Specific staining is seen in the zona glomerulosa (arrow). showing expression (dark stain) in the zona glomerulosa (arrow). Scale bars (A, B, E and F) 50 mm or (C and D) 100 mm. (B) Sense probe. Scale bar 100 mm.

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Figure 4 Real-time PCR analysis of EphA2 receptor transcription in control and treated animals. Low sodium (Low NaC), captopril and ACTH treatment caused a significant change in the level of EphA2 mRNA in adrenal glands (betamethasone was without effect) thus confirming the close relationship between EphA2 and the glomerulosa phenotype. Values are meansGS.E.M. of triplicate determinations normalised against GAPDH values and expressed as a percentage of control. **P!0.005; ***P!0.0001, unpaired t-test (nZ3–7). In these studies, controls were untreated for the ACTH, captopril and betamethasone experiments, and fed a flour diet supplemented with 1% NaCl for the low-sodium diet animals. The data were normalised against the appropriate controls, then Figure 5 PCR analysis of ephrin expression in the rat adrenal gland. combined for the purpose of illustration. (A) Expression of ephrinA and ephrinB ligands. Parallel PCRs were performed on samples from cDNA reactions performed in the C K that we postulate is required for an understanding of the presence ( ) or absence ( ) of reverse transcriptase. Reverse transcription-free samples acted as negative controls. Size markers regulation of adrenocortical zonation (Vinson & Ho 1998, are Hyperladder 1 (Bioline, London, UK). The 200 bp marker is Vinson 2003). indicated (arrow). The predicted 100–150 bp products were The present real-time PCR results show clearly that mRNA obtained for each primer pair. (B) Real-time RT-PCR analysis of coding for a wide range of EphRs are transcribed in the rat Ephrin A1 transcription in control and treated animals. Low sodium (Low NaC), captopril and ACTH treatment caused a significant adrenal gland (Fig. 1). Todetermine which of these are relevant reduction in the level of Ephrin A1 mRNA in adrenal glands to zonation, glands were taken from animals subjected to (betamethasone was without significant effect, P!0.08). Values are different treatments known to affect specifically the glomer- meansGS.E.M. of triplicate determinations normalised against ulosa/fasciculata phenotypes in the rat. Thus, a low-sodium GAPDH values and expressed as a percentage of control, as shown in Fig. 4.**P!0.01, unpaired t-test (nZ5–8). diet, which provokes angiotensin II generation, is widely used to stimulate the zona glomerulosa and aldosterone secretion in EphA2 expression consistent with the view that EphA2 (Aguilera & Catt 1983, Lehoux et al.1997, Raza et al.2005), reflects the expression of the zona glomerulosa phenotype and while the ACE inhibitor captopril suppresses it (McEwan et al. function: ACTH treatment, which decreases the glomerulosa 1996, van Kats et al.2005). Similarly, ACTH treatment phenotype, also decreased EphA2 expression whereas beta- suppresses aldosterone secretion, and quickly initiates the methasone, which specifically affects the fasciculata/reticularis, transformation of glomerulosa into fasciculata cells (Aguilera but not the glomerulosa, had no effect on EphA2. That these et al.1981, Pudney et al.1984, Abayasekara et al.1989): EphRs are indeed functional is suggested by the further finding prolonged treatment may result in the elimination of the that mRNA coding for ephrin A1, considered to be the most glomerulosa altogether (Vinson 2003). Betamethasone, by important ligand for the EphA2 receptor, is also transcribed in contrast, suppresses circulating ACTH, and hence fasciculata the rat adrenal, although in this case its transcription, though function and glucocorticoid secretion (Buckingham & Hodges affected by the various treatments, is enigmatic. Its lack of 1976, Cam & Bassett 1983, Raza et al.2005). response to betamethasone and its decrease following ACTH A clear relationship to the expected phenotypic changes in treatment reflects the changes seen with EphA2, but in adrenocortical cells that are brought about by the various contrast to the decreases in response to both a low-sodium diet treatments was revealed by EphA2 receptor mRNA, which and captopril were not clearly related to physiology (Fig. 5). ISH showed was exclusively limited to the zona glomerulosa, Failure to respond to glomerulosa-specific treatments in a as also is EphA receptor protein (Fig. 2). The transcription of predictable manner clearly does not exclude other EphR EphA2 receptor mRNA closely followed the expected subtypes from a glomerulosa site, and the transcription of glomerulosa phenotype: it was enhanced by a low-sodium mRNA coding for the A3 subtype also mostly occurs in the diet, but reduced by captopril treatment (Fig. 4). Furthermore, glomerulosa (Fig. 3), although unaffected by a low-sodium diet treatment with ACTH or betamethasone also induced changes or captopril (data not shown). www.endocrinology-journals.org Journal of Endocrinology (2008) 198, 185–191

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