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of Castilla y León, University of Salamanca, Spain; 2Department of Human Anatomy and Histology, Faculty of Medicine,. University of Salamanca, Spain; 3Joslin ...
Pituitary 5: 5–10, 2002 C 2003 Kluwer Academic Publishers. Manufactured in The Netherlands. 

Expression of Aromatase P450 is Increased in Spontaneous Prolactinomas of Aged Rats Jos´e Carretero 1,2 , Deborah Jane Burks 1,3 , Gabriel 2 2 , Manuel Rubio 1,2 , Elena Hernandez , Vazquez ´ ´ 1 1 ,2 Pilar Bodego , and Ricardo Vazquez ´ 1 Laboratory

of Neuroendocrinology, Institute for Neuroscience of Castilla y Leon, ´ University of Salamanca, Spain; 2 Department of Human Anatomy and Histology, Faculty of Medicine, University of Salamanca, Spain; 3 Joslin Diabetes Center, Harvard University, Boston, MA, USA

Abstract. We have recently reported the presence of aromatase P450 in the rat hypophysis. This enzyme is responsible for the aromatization of testosterone to estradiol. Since the induction of prolactinomas has been demonstrated in the rat following chronic treatment with estradiol, the aim of the present study was to analyze whether a relationship exists between the presence of pituitary aromatase and the appearance of spontaneous prolactinomas in aged rats. Of a series of 90 adenomas studied, 53% showed prolactin immunoreactive cells and were classified as prolactinomas; only 33% of the adenomas were pure prolactinomas and the other 20% were multi-hormonal protactinomas. Moreover, 60% of the adenomas were aromatase-positive tumors. Interestingly, 100% of the pure prolactinomas were aromatase-positive while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Western blotting with anti-aromatase antibodies revealed a 3.8-fold increase in expression of aromatase in pituitary tumors as compared to normal rat pituitary gland. Double immunohistochemical labeling detected the coexistence of prolactin and aromatase P450 in prolactinoma cells. ACTH- and LH-positive adenomas were considered as controls; only multi-hormonal ACTH and LH tumors display aromatase-positive cells and all of these also contained prolactin-positive cells. Our results demonstrate for the first time that aromatase is expressed in pituitary adenomas and that it is related to the functional nature of the tumor, especially in the case of pure prolactinomas, suggesting the possibility that an abnormally high conversion of testosterone into estradiol in pituitary cells may contribute to the genesis of spontaneous prolactinomas in aged rats. Key Words. aromatase-P450, immunohistochemistry, prolactinoma, hypophysis

Introduction Pituitary tumors arising from adenohypophyseal cells are the most common adult intracranial pathology, representing 10% of all brain tumors [1]. The majority are true clonal neoplasms rather than hyperplasias resulting from increased hypothalamic drive [2], although the mechanisms underlying the genesis of pituitary tumors remain poorly understood. It is well known that spon-

taneous pituitary adenomas are frequent in aged rats [3–6] and prolactinomas represent the most common type, 43% of pituitary adenomas in this species [5]. Several factors, such as estradiol, have been implicated in the genesis of pituitary adenomas, especially prolactinomas and VIPomas, following chronic treatment with the steroid [7–10]. Of the two pathways through which androgens are metabolized-reduction and aromatization- the latter depends on the presence of aromatase P450, which belongs to the family of cytochrome P450. We have recently reported that the immunohistochemical expression of aromatase P450 in the rat pituitary gland differs from males to females [11]. Based on these observations, we hypothesized that the aromatization of testosterone to estradiol in pituitary cells might be related to the genesis of spontaneous pituitary adenomas. Thus, the aim of the present study was to determine whether differences in the expression of aromatase exist in the pituitary glands of aged female rats with or without adenomas, particularly in spontaneous pituitary prolactinomas induced with age.

Materials and Methods Animals One hundred and fifty-five female Wistar rats of 24 months were surveyed. After necropsy, the presence of spontaneous pituitary adenomas was confirmed in 90. The animals were maintained from birth under standard housing conditions. They were sacrificed between 10.00 and 11.00 h under anesthesia with sodium pentothal. Immediately afterwards, the skulls were opened and the pituitaries, with or without tumors, were carefully removed and fixed by immersion in 4% paraformaldehyde in phosphate buffer saline (PBS: 0.1 M, pH 7.4, plus 0.8% NaCl) for 24 h at 4◦ C and then washed in PBS Address correspondence to: J. Carretero, Instituto de Neurociencias de Castilla y Leon, ´ Facultad de Medicina, Avda. Alfonso X el Sabio, s/n, E-37007 Salamanca, Spain. Tel: +34 (9)23 294546; Fax: +34 (9)23 294559; E-mail: [email protected]

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for 24 h. Prior to fixation, the non-tumoral pituitaries from 5 female rats and a portion of each pituitary tumor were processed for Western blot. The fixed glands were dehydrated in ethanol and embedded in paraffin to obtain 5 µm serial sections for immunohistochemical study.

Immunohistochemistry A battery of immunohistochemical tests was performed on each pituitary to classify the tumors, when present. Analysis was based on the following markers: Aromatase, Prolactin, LH, FSH, GH, TSH and ACTH. After endogenous peroxidase had been blocked by incubation in a 0.25% solution of H2 O2 in methanol and cross reactions of the second antibody had been R , diluted 1:30), secblocked with goat serum (Dako tions were incubated for 24 h at 4◦ C in a humid chamber with one of the following specific antibodies: anti-aromatase F-72 polyclonal serum (kindly provided by Prof. Hutchison, diluted 1:500) or anti-aromatase R ; diluted 1:500), anti-Prolactin Rb-871 (Sigma Genosys R rabbit polyclonal serum (Dako , diluted 1:800), anti-CH R diluted 1:900), anti-LH rabbit polyclonal serum (Dako R , diluted 1:1000), antirabbit polyclonal serum (Dako R , diluted 1:800), FSH rabbit polyclonal serum (Dako R , diluted anti-ACTH rabbit polyclonal serum (Dako R , 1:1000) and anti-TSH rabbit polyclonal serum (Dako diluted 1:1000). The reaction was allowed to progress by incubation for 40 min at room temperature with R , digoat anti-rabbit IgG biotinylated serum (Dako luted 1:100) and later with incubation for 35 min at room temperature with Streptoavidin-Peroxidase R , diluted 1:400). The reaction was de(ATIQ-Biosystems R , veloped with 3,3 -diaminobenzidine (0.025 M, Sigma in 0.05 M Tris-HC1 buffer, pH 7.4) to which 0.03% H2 O2 had been added. The specificity of the antiaromatase P450 serum used in the present study was confirmed by replacing the primary anti-serum with nonimmune rabbit or mouse serum and by pre-absorption of the antiserum. For the preabsorption test of antiaromatase Rb-871 polyclonal antibody, diluted antiaromatase Rb-871 serum (1:500) was pre-absorbed (24 h at 4◦ C) with the synthetic peptide sequence C-EIIFRHIFNTPFLQC corresponding to residues 489503 of Hickey’s isoform [12] of rat cytochrome P450arom (50 µg peptide/ml antibody solution), which was the antigen used in generation of the rabbit Rb871 polyclonal anti-aromatase serum. Subsequently, the immunohistochemical reaction was developed and using either test of specificity, no reactivity was detected. The tumors were classified on the basis of whether they showed any internal reaction to the marker analyzed. In the case of cells reactive to hormones, only tumors showing a density of reactive cells greater than the percentages established for normal tissue in the studies of Dada et al. [13] were considered positive.

Generation of anti-aromatase antibodies and Western blotting The F-72 antibody against aromatase P450 was kindly provided by Prof. Hutchison. Anti-aromatase F-72 serum was obtained against the peptide sequence C-EIIFSPRNSDKYLQQ corresponding to residues 488502 of mouse cytochrome P450arom and having 60% homology with residues 488-496 of the rat cytochrome P450arom [14]. The specificity of this antiserum was checked previously by immunohistochemistry, immunosuppression and Western blot by Beyer et al. [15–17]. Rb-871 anti-aromatase P450 antibody was obtained via a two-week immunization protocol and bleedings were performed in alternative weeks by Sigma R . For immunization, the peptide sequence CGenoSys EIIFRHIFNTPFLQC corresponding to residues 489-503 of rat cytochrome P450arom of Hickey’s isoform [12] was coupled to KLH / MBS carrier protein. For Western blotting, pituitary glands were dissected from adult rats and frozen immediately. Tissues were later disrupted by homogenization in lysis buffer (137 mM NaCl, 10 mM Tris pH 7.4, 10% glycerol and 1% Tx-100 containing a cocktail of protease inhibitors). Insoluble material was removed from lysates by centrifugation at 10,000 rpm for 10 min. Protein concentrations were determined using a standard Bradford assay. 50 µg of total protein from each rat sample was separated by 10% SDSPAGE. Following electrophoresis proteins were transferred to nitrocellulose and then blocked for 1 hr with 5% non-fat dry milk in PBS. Nitrocellulose membranes were then incubated overnight with either pre-immune serum or anti-aromatase antibodies diluted 1:500. Blots were subjected to 3 × 15 min washes with PBS and then incubated for 1 hr with HRP-labeled secondary antibodies (1:10,000 in PBS). Following extensive washing, blots R ). Average exposure were revealed by ECL (Amersham time was 2 min. Densitometric analysis was performed ( Molecular Dynamics) to assess relative levels of aromatase expression.

Results Aromatase expression in normal (non-tumorous), aged pituitary gland In rats of 24 months of age where adenomas were not detected in the hypophysis, aromatase-immunoreactive cells in the pituitary glands were either absent or scarce and displayed very weak immunoreactivity (Fig. 1(a)). Characterization of spontaneous pituitary adenomas Of the 90 adenomas studied, 83% had prolactinimmunoreactive cells. Of these, 40% (33% of the total) expressed only prolactin and therefore, were classified as pure prolactinomas, while 60% (50% of the total) reacted to more than two classic pituitary hormones. Of samples immunoreactive to prolactin, only 20% of the

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Fig. 1. (a) Micrograph demonstrating the absence of aromatase immunoreactivity in non-tumoral pituitary glands of aged rats (×450). (b) Panoramic view of an aromatase-immunoreactive pituitary microadenoma (×150). (c) Micrograph of aromatase-immunoreactivity in a solid pituitary adenoma (×750). (d) Micrograph showing aromatase-immunoreactivity in a hemorrhagic pituitary adenoma (×450). (e) Micrograph showing the different types of cells reactive to aromatase: glandular (arrow) and endothelial (arrowhead) (×750).

tumors displaying prolactin-immunoreactive cells were considered multi-hormonal prolactinomas (16% of the total), such that 53% of the tumors in our series were considered prolactinomas. Of the adenomas studied, a high percentage (60%) were aromatase-immunoreactive. No correlation was found between the pathological type of the adenomas and aromatase expression. Thus, the enzyme was detected in solid, hemorrhagic and cystic tumors, as well as in micro-adenomas (Fig. 1(b) and (c), but all were tumors derived from pars distalis. The aromatase immunoreactivity was noted mainly in the cytoplasm, although some cells displayed reactive nuclei. Additionally, aromatase expression was observed more frequently in glandular cells, although some tumors displayed reactivity in both glandular (arrow in

Fig. 1(d)) and stromal cells, such as endothelial cells (arrowhead in Fig. 1(d)).

Relationship between aromatase expression and prolactinomas In the examination of prolactinomas, we noted that 86% exhibited immunoreactivity for aromatase. Strikingly, 100% of the pure prolactinomas were aromatase positive (Fig. 2(a) and (b) while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Moreover, considering all the tumors positive for aromatase, 72% were prolactinomas (55% pure and 17% multiple). Using double immunolabeling of prolactinomas, coexistence of aromatase and prolactin within the same cells was observed (arrows in Fig. 2).

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tained prolactin-positive cells. Pure LH tumors comprised only 3% of total adenomas whereas 32% of multihormonal adenomas contained LH-positive cells. 73% of LH multi-hormonal adenomas were aromatase-positive and similar to our studies of ACTH multi-hormonal tumors, 100% of these tumors displayed prolactin immunoreactivity. No pure LH adenomas stained positive for aromatase.

Discussion

Fig. 2. Micrographs showing immunoreactivity to aromatase (a) and prolactin (b) in a pure, spontaneous prolactinoma. Note the co-expression of the enzyme and the hormone in the same cell (arrows) (×450).

To further define aromatase expression in pituitary tumors, Western blot analyses of pituitary samples were performed. Using anti-aromatase antibodies, Western blotting demonstrated the existence of a protein of approximately 50 Kda in tumoral and normal pituitary lysates (Fig. 3(A)). Pre-absorption of the anti-aromatase antiserum with the peptide used as antigen abolished detection of the 50 Kda band, suggesting specificity of the antibody for aromatase. Densitometric analysis of the immunoblots (Fig. 3(B)) revealed a 3.8-fold higher level of aromatase expression in tumoral samples as compared to normal controls. As controls, ACTH and LH immunoreactive adenomas were studied. 36% of adenomas contained ACTH positive cells. However, only 6% of adenomas were pure ACTH-adenomas and all these appeared in the pars intermedia and thus, were not included for analysis. Considering only tumors from pars distalis, 14% of pituitary adenomas were characterized as ACTH multi-hormonal adenomas. 78% of ACTH-multi-hormonal adenomas were aromatase positive adenomas; however, 100% of these ACTH- and aromatase-positive tumors also con-

The existence of pituitary adenomas was reported as early as the beginning of 20th century [18]. However, because the cells involved in pituitary adenoma formation may be derived from either monoclonal or polyclonal expansions and because they have phenotypes that are not only difficult to classify but also change during tumor development, the pathogenesis of pituitary tumors remains a subject of speculation and debate. The incidence of prolactinomas in aged rats is high; in some studies it reaches 80% in old Sprague-Dawley rats [19,20]. As the pathogenesis of pituitary adenomas suggests multi-factoral mechanisms for their genesis, different theories have been invoked to explain the molecular basis of these tumors including excessive trophic hormone action, receptor abnormalities, malfunctions of G proteins, alterations in protein kinases, local production of growth factors, anomalous vascular supply, maturation of tumor suppressor genes, and failure of programmed cell death [21]. Previous observations suggest failure of the hypothalamic inhibitory regulation of prolactin cells and their secretion [22]. Vascular alterations in the portal system have also been invoked to account for the appearance of prolactinomas. Depending on the duration of exposure, chronic treatment with estradiol induces the appearance of prolactinomas [23–26]. The fact that some strains of rats are not susceptible to the estradiol-induced appearance of prolactinomas suggests there may be an additional mitogenic factor or alterations in the expression of certain genes, such as the p53 anti-tumor protein associated with heterozygous mutations of the retinoblastoma gene [27–30]. Interestingly, recent studies from Heaney and colleagues [31] have demonstrated that prolactinsecreting tumors contain very high levels of the tumorigenic protein PTTG (pituitory tumor transforming gene). PTTG expressing cells display increased secretion of FGF and accelerated angiogenesis. Expression of PTTG is regulated by estrogen and this protein has been demonstrated to exert a direct role in early lactotroph transformation [31]. Additionally and/or alternatively, as is demonstrated in the current work, the appearance of such tumors could be related to the up-regulation of aromatase expression which occurs with age and changes in steroid levels in rats. In a previous study [11], we reported that aromatase expression in the pituitary gland is low in female adult rats. The present study demonstrates similar findings

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Fig. 3. (a) Western blots from tumorous (T) and non-tumorous (NT) pituitaries using different antibodies against aromatase (Rb-851 y F-72). After pre-absorption of anti-serum Rb-851 with antigen peptide, immunoreactive bands were not detected. (b)- Densitometric analysis of aromatase expression. Western blots were subjected to densitometry (Molecular Dynamics) and aromatase bands were assigned relative values.

in old female rats. This observation could reflect the natural evolution of the enzyme in response to deficits or declines in gonadal function in accordance with a previous study [32]. The fact that a very high percentage of spontaneous adenomas express aromatase, as reported by the present study, suggests that expression of the enzyme is closely linked to tumor induction, development or maintenance. Moreover, the strong relationship observed between aromatase expression and the appearance of pure prolactinomas implicates a specific role for this enzyme in genesis of these tumors. Administered chronically, estradiol induces the development of prolactinomas. Therefore, it would not be unreasonable to surmise that an abnormally high conversion of testosterone into estradiol due to the action of aromatase in pituitary cells would have the same consequences in aged rats as exogenous treatment with the steroid. Non-active isoforms of aromatase in testis have been detected by immunohistochemistry but the biological relevance of these isoforms is unknown [14]. The results obtained by Western blot in the present study demonstrate that aromatase in the pituitary gland is a 50 kDa protein, most liekly representing the intact, active aromatase. No fragments of the enzyme were detected by immunoblotting of pituitary lysates.

In summary, our results demonstrate a high expression of aromatase in spontaneous pituitary adenomas and suggest that aromatase may be involved in the genesis of spontaneous prolactinomas in aged rats.

Acknowledgments The authors wish to thank Prof. J.B. Hutchison for kindly supplying the F-72 anti-aromatase serum. This work was supported by the BIOMED 1 program of the EU. No. BMH1-CT94-1536, the European Social Fund and J. Castilla y Leon ´ program No. SA/58/98 and the FIS Spanish program No. 99/1187.

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