321 Expression of 11-hydroxylase and synthase genes in the rat brain

S M MacKenzie1, C J Clark1, R Fraser1, C E Gómez-Sánchez2, J M C Connell1 and E Davies1 1MRC Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, UK 2Department of Internal Medicine, University of Missouri–Columbia and the Harry S Truman Memorial Veterans Hospital, Missouri 65201–5297, USA (Requests for offprints should be addressed toSMMacKenzie, MRC Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow G11 6NT, UK; Email: [email protected])

ABSTRACT The terminal stages of and aldosterone immunolocalised in paraffin-embedded rat adrenal production in the human are catalysed and brain sections using mouse monoclonal anti- by the 11-hydroxylase and aldosterone bodies. Negative controls utilised a mouse mono- synthase, which are encoded by the CYP11B1 and clonal antibody raised against a non-mammalian CYP11B2 genes respectively. Recent studies have epitope. In the brain, 11-hydroxylase and aldoster- suggested that aldosterone and cortisol are also one synthase were detected in the cerebellum, made in other tissues such as the brain, heart and especially the Purkinje cells, as well as the vascular system and may play a role in cardiovascu- hippocampus. The specificities of the 11- lar homeostasis. The aim of this study was to hydroxylase and aldosterone synthase antibodies confirm the presence of these enzymes and localise were confirmed by positive immunostaining of the them precisely in the rat brain. relevant regions of the . Reverse transcription-polymerase chain reaction This is the first direct evidence that (RT-PCR)/Southern blotting confirmed transcrip- hydroxylases involved in the final stages of tion of CYP11B1 and CYP11B2 in whole brain biosynthesis are present in specific and hypothalamus minces from Wistar–Kyoto rats. regions of the central nervous system. 11-Hydroxylase and aldosterone synthase were Journal of Molecular Endocrinology (2000) 24, 321–328

INTRODUCTION 11-Hydroxylase is localised in the zona fasciculata and is regulated by adrenocorticotrophin (ACTH). Synthesis and secretion of mineralocorticoids and Aldosterone synthase is unique to the zona glucocorticoids from occurs in the glomerulosa and is regulated by the – adrenal cortex, with the initial step, the conversion angiotensin system (RAS) (Oertle & Müller 1993). of cholesterol to , performed by the The genes encoding 11-hydroxylase (CYP11B1) CYP11A1 gene product, the side-chain cleavage and aldosterone synthase (CYP11B2) have a high . The major glucocorticoid in man is percentage of sequence identity (Imai et al. 1990, cortisol; in the rat, it is . Aldosterone Nomura et al. 1993, Zhang & Miller 1996). is the major mineralocorticoid in both species. It is Both aldosterone and cortisol (or corticosterone) synthesised in the of the adrenal have important effects on balance and cortex while cortisol or corticosterone are made blood pressure by their actions on the and mainly in the zona fasciculata (White et al. 1992, the vasculature (Fraser et al. 1989). In addition Vinson et al. 1995). The final steps of cortisol and to these systemic effects, however, it is clear that aldosterone biosynthesis are catalysed by 11- they exert significant central effects. Three types hydroxylase and aldosterone synthase respectively. of evidence exist. First, direct intracerebral

Journal of Molecular Endocrinology (2000) 24, 321–328 Online version via http://www.endocrinology.org 0952–5041/00/024–321  2000 Society for Endocrinology Printed in Great Britain

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 1. Oligonucleotide primer sequences used for PCR amplification of cDNA

Length of product Primer Sequence (5–3) (bp) CYP11B1/2/3 Upstream AAC TCC GTG GCC TGA GAC G 342 Downstream GCT GTG TGG TGG ACT TGA AC Adrenodoxin Upstream GAC TCT CTG CTA GAT GTT GTG ATT 289 Downstream ATT CTT GCT CAT GTC AAC AGA CTG TCG CYP11A1 Upstream CAA CAT CAC AGA GAT GCT GGC AGG 583 Downstream CTC AGG CAT CAG GAT GAG GTT GAA

administration of doses too small to affect systemic opposed to gene transcription, and more precise concentrations elicit effects which may differ from information on their location within the brain have their classical systemic effects. For example, small been obtained by immunohistochemistry. doses of aldosterone raise blood pressure in the rat without the accompanying changes in extracellular sodium or potassium concentrations (Gómez- MATERIALS AND METHODS Sánchez 1997) characteristic of systemic admin- istration. Secondly, the brain is abundantly supplied RNA isolation with corticosteroid receptors. The glucocorticoid receptor is widely distributed but the mineralo- Tissues from adult female Wistar–Kyoto rats fed corticoid receptor is particularly concentrated in the on normal diets were snap frozen in liquid nitrogen hippocampus and hypothalamus (De Kloet et al. for subsequent RNA extraction. Total RNA was 1998). Thirdly, there is increasing evidence to extracted from these tissues according to the suggest that can be synthesised in standard RNAzol B reagent protocol (Biogenesis the brain. Both CYP11B1 and CYP11B2 appear Ltd, Poole, Dorset, UK). The quality and con- to be transcribed in brain tissue (Strömstedt & centration of the resulting RNA was confirmed by Waterman 1995). There is also some evidence that spectrophotometry and electrophoresis on ethidium brain tissue in vitro converts precursors to end bromide-stained agarose gels. products (Gómez-Sánchez et al. 1997) although de novo synthesis from cholesterol has yet to be RT-PCR demonstrated. Rates of synthesis are likely to be small in comparison to those of the adrenal cortex. Oligonucleotide primers (Oswel, Southampton, However, since the brain is highly sensitive to Hants, UK) homologous to parts of the CYP11B1/ corticosteroids (see above), low level, local synthesis B2, adrenodoxin and CYP11A1 genes were pro- may be physiologically important. duced (Table 1). RT-PCR amplification was We investigated the transcription of CYP11B1, performed using the GeneAmp RNA PCR kit CYP11B2 and CYP11A1 in several rat tissues. (Perkin Elmer, Warrington, UK) and oligo-d(T) Evidence of CYP11A1 expression in extra-adrenal primers. cDNA was transcribed according to the tissues would support the de novo formation of standard kit protocol. Amplification of CYP11A1 corticosteroids. In addition, we also tested for the and adrenodoxin were performed under identical transcription of adrenodoxin, a cofactor required conditions, following the method of Strömstedt & by mitochondrial P450 enzymes such Waterman (1995). There was a final extension step as aldosterone synthase, 11-hydroxylase and the of 7 min at 74 C, after Nomura et al. (1993). side-chain cleavage enzyme. Controls had reverse transcriptase enzyme omitted, Evidence of corticosteroid synthesis in the rat or water substituted for RNA. brain has so far been obtained by reverse transcription-PCR (RT-PCR) or by conversion of steroid precursors to end products in tissue Southern blotting homogenates. To assess its potential role, it is RT-PCR products were run on 2% agarose gels and necessary to define more precisely the site(s) of Southern blotted to Hybond-N+ membrane synthesis. With this in mind, evidence of aldoster- (Amersham Life Science, Amersham, Bucks, UK). one synthase and 11-hydroxylase production, as CYP11B1 and CYP11B2 transcript products were

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Downloaded from Bioscientifica.com at 09/30/2021 04:06:01PM via free access Brain 11-hydroxylase and aldosterone synthase ·    and others 323 distinguished by hybridising with radiolabelled performed using the DAKO Catalysed Signal oligonucleotide probes. The CYP11B1-specific Amplification (CSA) System Peroxidase Kit oligonucleotide was 5-TAA ACA TTC AGT CCA (DAKO Ltd, Cambridge, Cambs, UK). Primary ATA-3; the CYP11B2-specific oligonucleotide was antibody was incubated overnight at 4 C. Im- 5-TGG ATG TCC AGC AAA GTC-3 (position munostaining was developed with 3,3- 556–574 bp, exon III). CYP11A1 (the gene for diaminobenzidine tetrahydrochloride (DAKO Ltd). P450scc) transcripts were identified using the Sections were then counterstained with filtered oligonucleotide 5-GGT GGA GTC TCA GTG modified Harris haematoxylin solution (Sigma- TCT CCT TGA TGC TGG CTT TGA G-3.An Aldrich Co. Ltd, Poole, Dorset, UK) for 5 min, adrenodoxin-specific radiolabelled oligonucleotide dehydrated, and mounted in DPX (BDH Labora- was not required as any resulting bands were clearly tory Supplies, Lutterworth, Leics, UK). Control visible on ethidium bromide-stained gels under UV sections were incubated with mouse IgG1 negative light. control culture supernatant (DAKO Ltd). In addition, specificity of the monoclonal antibodies was confirmed by incubating them with an excess of Monoclonal antibody preparation their respective antigenic polypeptides (Alta Bio- Synthetic peptides corresponding to hydrophilic science, Birmingham, UK), thus quenching any areas of aldosterone synthase and 11-hydroxylase antibody binding. exhibiting minimal were prepared by Research Genetics Inc. (Huntsville, AL, USA). The aldosterone synthase monoclonal antibody was RESULTS generated by immunising CD-1 mice, as described by Tam (1988), with the multiple antigenic peptide RT-PCR MAP-KVRQNARGSLTMDVQQ corresponding CYP11B1 (Fig. 1a) and B2 (Fig. 1b) were to residues 175–190. The 11-hydroxylase mono- transcribed in the adrenal gland, whole brain and clonal antibody was generated in Swiss–Webster hypothalamus. There was no evidence of expression mice immunised with the peptide KNVYRE- in the kidney, liver or heart. All the tissues tested LAEGRQQS corresponding to residues 272–285, were positive for adrenodoxin transcripts (Fig. 1c). and conjugated to chicken serum albumin. The There was also evidence of CYP11A1 transcription spleens of the mice with the highest titre were in brain tissue (Fig. 1d). All control reactions were fused to an SP-2 myeloma cell line and the clones negative. selected for their ability to bind immobilised sonicated zona glomerulosa mitochondria from rats on a low sodium diet. Both antibodies were of the Immunohistochemistry IgG1 type. The specificities of the anti-11-hydroxylase and anti-aldosterone synthase antibodies were con- Tissue preparation for immunohistochemistry firmed by immunohistochemistry in rat adrenal gland sections (Fig. 2). The anti-aldosterone Brains and adrenal glands were removed from synthase antibody produced staining only in the adult female Wistar–Kyoto rats immediately zona glomerulosa whereas the anti-11-hydroxylase after lethal injection and fixed for 24 h in 4% antibody bound to the zona fasciculata and zona (w/v) paraformaldehyde solution before being reticularis but not to the zona glomerulosa. There embedded to form tissue blocks. Sections 5 µm were also projections of cells stained for 11- thick were prepared from these, mounted on hydroxylase into the adrenal medulla. 3-aminopropyltriethoxysilane-treated slides and In the brain, immunostaining of both 11- air-dried. hydroxylase and aldosterone synthase occurred in the cerebellar cortex (Fig. 3), particularly within the Purkinje cells. 11-Hydroxylase and aldosterone Immunohistochemistry synthase were also detected in the hippocampus, Tissue sections were deparaffinised with xylene and with greatest intensity in the dentate gyrus and CA3 rehydrated through graded alcohols before being cells (Fig. 4). No evidence of translation could be rinsed in deionised water and Tris-buffered saline found in the hypothalamus. Immunostaining could (TBS). The sections were then subjected to not be seen in control sections. Positive immuno- microwave antigen-retrieval in 0·01 M citrate staining was quenched by prior incubation with buffer, pH 6·0 for 10 min. Subsequent steps were excess antigenic peptide. www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 321–328

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 1. RT-PCR of RNAs from various tissues in the presence (+) and absence (–) of reverse transcriptase. (a) CYP11B1, (b) CYP11B2, (c) adrenodoxin, (d) CYP11A1. Products were detected by Southern blotting using specific, end-labelled radioactive oligonucleotides, except for adrenodoxin transcripts which were detected by ethidium bromide staining under UV light.

 2. Immunohistochemical localisation of enzymes in rat adrenal tissue. (a) Staining for 11-hydroxylase in the zona fasciculata, zona reticularis and adrenal medulla (bar=500 µm), (b) staining for 11-hydroxylase in the zona fasciculata (bar=50 µm), (c) staining for aldosterone synthase in the zona glomerulosa (bar=50 µm), (d) absence of staining in control section incubated with antibody raised against non-mammalian epitope (bar=50 µm).

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 3. Immunohistochemical localisation of enzymes in rat cerebellum. (a) Staining for 11- hydroxylase (bar=500 µm), (b) detail showing 11-hydroxylase staining in Purkinje cells (bar=50 µm), (c) staining for aldosterone synthase (bar=500 µm), (d) detail showing aldosterone synthase staining in Purkinje cells (bar=50 µm), (e) absence of staining in control section incubated with antibody raised against non-mammalian epitope (bar=500 µm), (f) detail showing Purkinje cells in control section (bar=50 µm).

DISCUSSION & Robel 1990). There is now increasing evidence to suggest that corticosteroid production, once Until recently, it was believed that steroid regarded as confined to the adrenal cortex, can also hormone biosynthesis takes place in a limited range occur in the brain and in vascular tissue (Baulieu & of tissues including the adrenal cortex, gonads Robel 1990, Strömstedt & Waterman 1995, Takeda and placenta. However, it is well known that et al. 1996, Gómez-Sánchez et al. 1997). In this neurosteroids such as pregnenolone, study, we have investigated the possibility of local and dehydroepiandrosterone (DHEA) are synthe- corticosteroid production in a number of tissues, sised in the central nervous system (CNS) (Baulieu but with particular emphasis on the CNS. RT-PCR www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 321–328

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 4. Immunohistochemical localisation of enzymes in rat hippocampus. (a) Staining for 11- hydroxylase (bar=500 µm), (b) detail showing 11-hydroxylase staining in CA3 cells (bar=50 µm), (c) staining for aldosterone synthase (bar=500 µm), (d) detail showing aldosterone synthase staining in CA3 cells (bar=50 µm), (e) absence of staining in control section incubated with antibody raised against non-mammalian epitope (bar=500 µm), (f) detail showing CA3 cells in control section (bar=50 µm).

was used to show transcription of CYP11B1 molecular weight of 52 000 (data not shown) (11-hydroxylase) and CYP11B2 (aldosterone syn- which approximates to the theoretical molecular thase). Immunohistochemical techniques in brain weight of the enzyme. Aldosterone synthase tissue, utilising specific monoclonal antibodies concentration is too low to be detected by this raised against the respective enzymes, provided firm method. evidence of translation. This approach was validated RT-PCR showed clearly that CYP11B1 and by the specificity of the antibodies in the adrenal CYP11B2 are transcribed in the brain. Some cortex. Furthermore, following SDS-PAGE and transcription could be attributed more precisely to immunoblotting, the anti-11-hydroxylase antibody the hypothalamus, which was analysed separately. identified a protein band in adrenal tissue with a This confirms previous work by other groups (see

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Downloaded from Bioscientifica.com at 09/30/2021 04:06:01PM via free access Brain 11-hydroxylase and aldosterone synthase ·    and others 327 above). In addition, we and others (Mellon & Our experiments show unequivocally that there is Deschepper 1993, Strömstedt & Waterman 1995, 11-hydroxylase-like and aldosterone synthase-like Gómez-Sánchez et al. 1997) have shown that the immunoreactivity in discrete areas of the rat CNS. genes encoding side-chain cleavage enzyme, 21- These include the hippocampus, in particular the hydroxylase and adrenodoxin are also present in dentate gyrus, and the cerebellum, especially the brain tissue. Indeed, adrenodoxin appears to be Purkinje cells. Furukawa et al. (1998) discovered a transcribed ubiquitously. These findings suggest similar distribution for the transcripts of other that the full complement of enzymes and co-factors enzymes involved in this pathway (StAR, P-450scc necessary for de novo production of corticosteroids and 3-HSD) using in situ hybridisation, and this from cholesterol is present in the brain. further supports the hypothesis that de novo The presence of gene transcripts does not provide production of corticosteroids can occur in certain unequivocal evidence that translation to a functional regions of the brain. protein occurs. However, other groups have shown Although the presence of the enzymes in the CNS that brain minces from intact and adrenalectomised is clear, their function and regulation are not. rats can synthesise corticosterone and aldosterone Glucocorticoid and mineralocorticoid receptors are from endogenous precursors and also from radio- abundant in the CNS, the latter especially so in the labelled 11-deoxycorticosterone (DOC) (Gómez- hippocampus, an area associated with memory and Sánchez et al. 1997). Production can be inhibited learning (Agarwal et al. 1993, De Kloet et al. 1998). by metyrapone, a relatively specific inhibitor of Activation of the central mineralocorticoid receptor 11-hydroxylase (Gower 1974). It is also possible raises blood pressure and alters thirst and salt that adrenal-derived precursors such as DOC will appetite (Gómez-Sánchez 1997). The effects on act as substrates for 11-hydroxylase and aldoster- blood pressure of mineralocorticoid administration one synthase in other tissues. However, in man this direct to the CNS have already been remarked seems unlikely given the low circulating levels of upon. DOC (low nM range) and the reported Km values Although the techniques employed here are, at (µM range) of the enzymes in question (Fardella best, semi-quantitative, it is beyond dispute that et al. 1996). the corticosteroidogenic enzymes in the brain are There are discrepancies between the data gained found at much lower levels than in the adrenal from RT-PCR and from immunohistochemistry. cortex. However, the resulting amounts of corti- For example, there was no evidence of the enzymes’ costeroid, even if low, could be capable of expression in the hypothalamus using immuno- significant physiological effects. Gómez-Sánchez histochemistry, despite suggestions from RT-PCR (1997) showed that intracerebroventricular admin- to the contrary. However, RT-PCR is a highly istration of very small doses of aldosterone to the sensitive technique and the levels of translated rat increased systemic blood pressure (see earlier). enzyme may be beyond immunohistochemistry’s This effect could be specifically inhibited by present detection capabilities. Another possibility is mineralocorticoid receptor antagonists. Thus, even that CYP11B1 and CYP11B2 are regulated at the small amounts produced locally could exert signifi- translational rather than the transcriptional level cant physiological effects via paracrine or autocrine (e.g. transcripts in certain tissues may be unstable actions at this site. Aldosterone produced locally and degrade quickly). Moreover, the dissection of may be implicated in vascular tone control in the rat the brain into specific regions before isolating RNA (Takeda et al. 1995, Takeda et al. 1997) and, in is technically demanding and the risk of cross- man, aldosterone administration has recently been contamination between brain regions is high. For shown to modulate baroreceptor function (Yee & this reason, RT-PCR is not as reliable for the Struthers 1998). localisation of expression as immunohistochemistry. Glucocorticoids have widespread actions on the These conflicting findings emphasise the impor- brain, and assist in the regulation of hypothalamic/ tance of following up RT-PCR studies with pituitary/adrenal function by interacting with confirmation of enzyme synthesis. glucocorticoid receptors in the hypothalamus and The second part of this study was designed to anterior pituitary. (The hippocampus is important localise aldosterone synthase and 11-hydroxylase here.) They could also have major effects on precisely within the tissue. Demonstration of the long-term cardiovascular homeostasis (Seckl 1997). expected distribution of aldosterone synthase in Increased CNS exposure to glucocorticoids during the zona glomerulosa and of 11-hydroxylase in the fetal development has lifelong effects on cardiovas- zonae fasciculata and reticularis using immuno- cular and metabolic function. Local CNS synthesis histochemistry, previously shown by Ogishima et al. of corticosteroids may provide another mechanism (1992), confirmed the performance of this method. to determine tissue glucocorticoid exposure at key www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 321–328

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developmental times. Deviations above or below Imai M, Shimada H, Okada Y, Matsushima-Hibiya Y, normal glucocorticoid levels in the hippocampus are Ogishima T & Ishimura Y 1990 Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in also known to change the function of hippocampal rat adrenal cortex. FEBS Letters 263 299–302. cells and even result in their apoptosis (Reagan & Mellon SH & Deschepper CF 1993 Neurosteroid biosynthesis: McEwen 1997). Local production in the rat genes for adrenal steroidogenic enzymes are expressed in the hippocampus may be involved in these processes. brain. Brain Research 629 283–292. Having confirmed that the corticosteroidogenic Nomura M, Morohashi K-I, Kirita S, Nonaka Y, Okamoto M, Nawata H & Omura T 1993 Three forms of rat CYP11B apparatus does occur locally, the control mech- genes: 11-hydroxylase gene, aldosterone synthase gene, and anisms regulating its expression in the CNS, a novel gene. Journal of Biochemistry 113 144–152. which may differ from those in the adrenal cortex, Oertle M & Müller J 1993 Two types of cytochrome P-45011 should be investigated. The clinical implications of in rat adrenals; separate regulation of . Molecular and Cellular Endocrinology 91 201–209. extra-adrenal corticosteroidogenesis remain to be Ogishima T, Suzuki H, Hata J-I, MitaniF&IshimuraY1992 explored. Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011 in rat adrenal cortex: histochemical basis for the functional zonation. Endocrinology ACKNOWLEDGEMENTS 130 2971–2977. Reagan LP & McEwen BS 1997 Controversies surrounding glucocorticoid-mediated cell death in the hippocampus. We thank Professor Jonathan R Seckl, Molecular Journal of Chemical Neuroanatomy 13 149–167. Medicine Centre, Western General Hospital, Seckl JR 1997 11-Hydroxysteroid dehydrogenase in the brain: Edinburgh for advice and Mr Niall Whyte, a novel regulator of glucocorticoid action? 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