ARTICLE |
Correspondence to: Mahlon D. Johnson, Div. of Neuropathology, Dept. of Pathology, C-3321, MCN, Vanderbilt U. Medical Center, Nashville, TN 37232-2561. E-mail: mahlon.johnson@mcmail.vanderbilt.edu
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Summary |
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Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas. (J Histochem Cytochem 50:15091515, 2002)
Key Words: adenohypophysis, pituitary adenoma, carboxypeptidase D (CPD), carboxypeptidase E (CPE), carboxypeptidase Z (CPZ), adrenocorticotroph, fibrous body, immunohistochemistry
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Introduction |
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SEVERAL PITUITARY HORMONES, including adrenocorticotropic hormone (ACTH), growth hormone (GH), prolactin (PRL), chromogranin A and B, and proenkephalin, are synthesized as larger precursors that are processed post-translationally and targeted to either the constitutive or regulated pathways (
CPE (enkephalin convertase, carboxypeptidase H; EC 3.4.17.10), is a 5060 kD protein and is involved in the biosynthesis of most neurotransmitters and peptide hormone. It was first isolated and cloned in humans in 1990 (
We have recently identified CPE in normal ACTH-synthesizing corticotrophs, GH-producing somatotrophs, and PRL-producing lactotrophs in the human adenohypophysis, suggesting that CPE may participate in prohormone processing to produce these hormones (
CPD was isolated and cloned in 1995 in bovine, 1997 in human, and 1998 in mouse tissues. It is a 180-kD protein that is more than three times larger than CPE (
CPZ, another novel carboxypeptidase, was cloned in human in 1997 and in 1998 in rat (
An alteration of prohormone processing has been implicated in the pathogenesis of some pituitary adenomas. Studies by
The findings presented here suggest significant CPE and CPZ expression in ACTH-, GH-, and PRL-producing pituitary adenomas, but only CPE in null cell-type adenomas. CPD immunoreactivity, which is restricted to ACTH-producing corticotrophs in the normal adenohypophysis, is also extensive in ACTH-, GH-, and PRL-secreting adenomas, suggesting induction of CPD expression in adenomas. This is the first study to demonstrate expression patterns of CPs in human pituitary adenomas.
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Materials and Methods |
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Human Pituitary Adenomas
Forty-eight cases of pituitary adenomas were included in this study. Among them, eight were GH-producing somatotroph cell adenomas, 11 were ACTH-producing corticotroph cell adenomas, 13 were PRL-producing lactotroph cell adenomas, three were luteinizing hormone/follicle stimulating hormone (LH/FSH)-producing gonadotroph cell adenomas, and 13 were null-cell adenomas. Adenomas were classified as "null-cell" adenomas if they exhibited no granular eosinophilic cytoplasm, chromogranin immunostaining, but no or rare LH and or FSH immunoreactivity (
Antibodies
Antibodies against CPE, CPD, and CPZ were gifts from Dr. Lloyd Fricker (Albert Einstein College of Medicine, Bronx, NY). A rabbit polyclonal antiserum raised against the C-terminal portion of CPE was used at a dilution of 1:1000; an antibody raised against the C-terminal portion of the CPD was used at 1:500. A rabbit polyclonal antibody raised against CPZ was used at a dilution of 1:100. The specificity of these antisera has been characterized previously by Western blotting analysis (
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Immunohistochemistry
Immunostaining was performed on a Ventana ES automated immunostainer (Ventana; Tucson, AZ). After trials of different pretreatments for each antibody, the most appropriate conditions for each antibody were chosen, as shown in Table 1. Deparaffinized sections were pretreated with a microwave retrieval system (Antigen Retrieval; Biogenex, San Ramon, CA) and were then incubated with the primary antibodies. The antigenantibody complexes were visualized by an ABC detection system using diaminobenzidine (DAB) as the chromogen. Normal human pituitary, known to express CPE, CPD, and CPZ, served as positive controls. In addition, sections of normal adrenal gland and placenta were used as a positive control (
To further evaluate the specificity of CPD immunoreactivity and possible nonspecific binding to keratin, keratin from human epidermis (Sigma) was resuspended in distilled H2O and added to Ventana diluent containing CPD antibody (1:500 dilution) at a saturating concentration of 200 µg/ml, then incubated at 37C for 32 min (the time and temperature at which antibody was incubated with the tissue sections). Then sections from a "sparsely granular" and typical somatotrophic cell adenoma were subjected to immunohistochemistry as described above.
Scoring for Immunostaining Intensity
The staining intensity and number of stained cells were examined independently by two reviewers (XF and MJ) and were scored as 0 for no significant staining, + for scattered or focal intense immunostaining, and ++ for intense immunostaining in approximately 50% or more of tumor cells.
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Results |
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Expression of CPs in ACTH-producing Corticotrophic Adenomas
CPE immunoreactivity was detected in 10/11 adenomas. CPD immunoreactivity, which was usually significant, was seen in 10/11. CPZ immunostaining was seen in 8/11 corticotrophic adenomas (Table 2; Fig 1).
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Expression of CPs in GH-producing Somatotrophic Cell Adenomas
Cam 5.2 immunoreactivity was dot-like and juxtanuclear, consistent with the pattern of "sparsely granular" adenomas, in two cases. Widespread Cam 5.2 immunoreactivity throughout the cytoplasm was seen in the remaining adenomas studied.
CPE immunoreactivity was detected in 5/8 adenomas. Extensive CPD immunoreactivity was seen in the adrenal gland and epithelium of placenta (positive controls), but no immunostaining was seen in the section of keratin-rich epidermoid carcinoma used as a control for nonspecific binding. In adenomas, CPD immunoreactivity was extensive, seen in 8/8 (Table 2; Fig 1). Moderate to strong juaxtanuclear dot-like immunoreactivity was seen in the two cases exhibiting dot-like Cam 5.2 immunostaining, although the extent of the immunoreactivity varied and often involved a larger zone than that seen with Cam 5.2. In the other adenomas, CPD immunoreactivity was juxtanuclear in a dot-like pattern, sometimes accompanied by perinuclear or cytoplasmic immunostaining. This contrasted with the largely diffuse Cam 5.2 immunostaining in these cases (Fig 1). Preincubation of CPD antibody with keratin had no effect on the distribution or intensity of CPD immunoreactivity in an apparent "sparsely granular" and typical adenoma. CPZ immunostaining was seen in 5/8 somatotrophic adenomas.
Expression of CPs in PLR-producing Lactotrophic Cell Adenomas
Twelve of 13 adenomas exhibited CPE immunoreactivity in tumor cells. The extent of immunoreactivity appeared similar to that in normal adenohypophyseal cells. CPD immunoreactivity, which was usually significant, was seen in 12/13; CPZ immunostaining was seen in 12/13 lactotrophic adenomas (Table 2; Fig 1).
Expression of CPs in LH/FSH-producing Gonadotrophic Cell Adenomas
One of three gonadotrophic cell adenomas exhibited focal CPE immunoreactivity. None of the tumors exhibited CPD immunostaining. In contrast, 2/3 exhibited significant CPZ immunostaining (Table 2).
Expression of CPs in Non-functioning Null-Cell Adenomas
CPE immunoreactivity was detected in 11/13 adenomas. Limited, scattered CPD immunoreactivity was seen in 3/13 adenomas. Six of 13 exhibited limited CPZ immunostaining (Table 2; Fig 1). In contrast, as reported previously, focal CPD and more widespread CPZ immunoreactivity was seen in normal tissue (Table 2).
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Discussion |
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In the present study, extensive CPE, CPD, and CPZ immunoreactivity was detected in ACTH-synthesizing corticotrophic cell adenomas, GH-producing somatotrophic cell adenomas, and PRL-producing lactotrophic cell adenomas, suggesting that they may participate in the processing of a number of precursor peptides in these tumors, including their characteristic hormones and the chromogranins. In a previous study we found extensive CPE and CPZ immunoreactivity in adenohypophyseal cells that secrete ACTH, GH, and PRL. Their expression in adenomas, which in most cases secreted increased amounts of these hormones, suggests that they play a role in possible prohormone processing of these hormones. We have previously found that CPD immunoreactivity in the normal adenohypophysis is largely confined to ACTH-producing corticotrophs (
Recent studies in CPE-deficient mice [Cpe(fat)/Cpe(fat)] indicated the existence of CPE-like carboxypeptidases, such as CPD and CPZ. Carboxypeptidase D is a potential candidate to carry out redundant processing functions of CPE based on comparative distribution studies in the rat central nervous system (
In the present study, a dot-like staining pattern was seen in the two "sparsely granular" GH-producing somatotrophic cell adenomas. This pattern, along with perinuclear and variable cytoplasmic immunoreactivity was observed in the other somatotroph cell adenomas. The subcellular structures recognized are not known but may represent juxtanuclear/perinuclear and cytoplasmic binding to secretory granules, including those associated with fibrous bodies. The fibrous body (FB), named by Cardell and Knighton in 1966, was first described by Racadot in 1964. Despite many subsequent studies, the functional roles of FBs are not established. They are spherical bodies consisting of intermediate filaments (cytokeratins), secretory granules, mitochondria, and lysosomes intermingled with the microfilaments. They localize adjacent to the nucleus in the Golgi region and have frequently been seen in sparsely granulated somatotrophic adenomas, somatolactotrophic adenomas, acidophilic stem-cell adenomas (
LH and FSH are synthesized in their final forms without precursors. Therefore, it is not surprising that LH- and FSH-producing gonadotrophic cell adenomas exhibit little CPE and CPD immunoreactivity. The presence of CPZ in these cells most likely reflects a role in processing other prohormones, such as chromogranin A and B, which are ubiqutiously present in anterior pituitary cells and are synthesized as prepro-chromogranin. After cleavage by PC1 and PC2, the carboxyl terminals of these prohormones are modified by carboxypeptidases such as CPE, CPD, and CPZ (
Null-cell adenomas are believed to arise from gonadotropin-producing stem cells with very low functional activity (
CPD and CPZ are involved in post-translational processing, not only in the normal pituitary gland but also in neoplastic pituitary gland, especially in POMC-, GH-, and PRL-producing adenomas.
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Acknowledgments |
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We wish to thank Ms Rebecca Meitus, Tracy Moss, and Mr Brent Weedman for technical assistance.
Received for publication December 31, 2001; accepted June 5, 2002.
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