Nitric oxide synthase in the human pituitary gland.

Nitric oxide (NO) is generated by the NO synthase family of isozymes, which is present in many mammalian cells. The constitutive NO synthase isozymes generate NO, which acts via signal transduction mechanisms in the regulation of many functions including vascular tone and blood pressure, and the inducible isozymes mediate immunological mechanisms by cytotoxic and cytostatic effects. To determine whether NO has a role in anterior pituitary cell function, immunohistochemistry and in situ hybridization analyses were used to study NO synthase expression in normal and neoplastic human pituitary tissues. Brain NO synthase was localized in the anterior pituitary in secretory and in folliculo-stellate cells and in the posterior pituitary. Pituitary adenomas had higher levels of brain NO synthase protein and mRNA compared with normal pituitaries. Endothelial NO synthase was also present in anterior and posterior pituitary cells and in endothelial cells of the pituitary. Immunoblotting studies with brain NO synthase antibodies detected a slowly migrating approximately 155-kd band and more rapidly migrating approximately 90-kd and approximately 60-kd bands. Endothelial NO synthase, but not macrophage NO synthase, was also detected in the pituitary by immunoblotting studies, confirming the immunohistochemical observations. These findings indicate that NO synthase is expressed in normal and neoplastic human pituitary tissues with increased levels of brain NO synthase protein and mRNA in adenomas compared with non-neoplastic pituitary cells and suggest that NO may play a regulatory role in hormone secretion in anterior pituitary cells.     

Analysis of the chromogranin A post-translational cleavage product pancreastatin and the prohormone convertases PC2 and PC3 in normal and neoplastic human pituitaries.

Several members of the chromogranin/secretogranin (Cg/Sg) family are post-translationally processed in neuroendocrine cells and tumors to smaller peptides, some of which are biologically active. For example, CgA is processed to pancreastatin, parastatin, and other peptides. We analyzed the distribution of pancreastatin and CgA proteins in normal and neoplastic pituitaries as well as the prohormone convertases PC2 and PC3/1 (PC3), the putative processing enzymes for the Cg/Sg family, in 35 pituitary adenomas and 4 non-neoplastic pituitaries by immunohistochemistry and immunoblotting with highly specific antisera. CgA and CgB mRNAs were also examined. Pancreastatin was present in all subtypes of pituitary tumors, although prolactin-secreting adenomas expressed this peptide less frequently than did other tumor types. CgA protein and CgA mRNA expression were also restricted in prolactin adenomas and in normal prolactin cells, as shown by combined in situ hybridization and immunostaining. The prohormone convertases PC2 and PC3 were present in pituitary tumors and in non-neoplastic pituitaries. Immunoblot analysis and immunostaining showed a principal approximately 69-kd PC3 band and a approximately 68-kd PC2 band. Adrenocorticotrophic hormone-secreting adenomas expressed mainly PC3 as determined by immunoblotting and immunohistochemistry, whereas all other adenoma groups expressed predominantly PC2. These results indicate that the enzymes capable of processing CgA and other members of the Cg/Sg family to peptides with biological activity such as pancreastatin are widely expressed in human pituitary adenomas and in non-neoplastic pituitaries, with adrenocorticotrophic hormone tumors expressing predominantly PC3 and other adenomas expressing mainly PC2. The infrequent expression of CgA protein and pancreastatin peptides in normal and neoplastic prolactin cells suggests a unique role of CgA in these tumors.     

Neural cell adhesion molecule (NCAM) in normal and neoplastic human pituitary tissues: Analysis by immunohistochemistry and in situ hybridization

The expression of the neural cell adhesion molecule (NCAM) in 6 normal human pituitaries and 25 pituitary adenomas was investigated by immunohistochemistry and in situ hybridization. NCAM protein and mRNA were present in all normal and neoplastic human pituitary tissues. There were tumor type-specific differences in the distribution of NCAM in various pituitary adenomas. Growth hormone adenomas and prolactin-producing adenomas usually expressed lower levels of NCAM, compared to null cell and gonadotroph adenomas. Adreno-corticotropic hormone adenomas expressed the highest levels of NCAM mRNA. Six freshly dissociated pituitary adenomas were cultured in serum-free medium for 7 days to analyze the regulation of NCAM mRNA by in situ hybridization. The lower levels of NCAM expression in growth hormone and prolactin adenomas were not present in cells cultured for 7 days in serum-free medium on extracellular matrix. Phorbol 12-myristate 13-acetate (PMA) stimulated NCAM mRNA expression in 5 of 6 tumors. Gonadotropin-releasing hormone and growth hormone-releasing hormone increased NCAM expression in some adenomas. This study demonstrates that there is a variable expression of NCAM in pituitary adenomas and that hypotha-lamic hormones and PMA can regulate NCAM mRNA levels in neoplastic pituitary cells.     

Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas.

Protein kinase C (PKC) is involved in the differentiation and growth regulation of a variety of tissues including anterior pituitary gland cells. To determine the distribution of PKC in different types of adenomas, PKC activity was analyzed in human pituitary tumors and the effects of hypothalamic hormone stimulation on PKC activity were examined in cultured adenoma cells. Gonadotroph (LH/FSH) and null cell adenomas had significantly higher levels of particulate, soluble, and total PKC activity compared with growth hormone (GH) adenomas (P < 0.05). Chronic stimulation of null cell adenomas with gonadotropin hormone-releasing hormone or of one GH adenoma with GH-releasing hormone for 7 days did not significantly alter total PKC activity in pituitary cells cultured in serum-free medium. Localization of the calcium-dependent PKC isozymes (alpha, beta and gamma) by immunohistochemistry and in situ hybridization revealed predominantly PKC alpha in all adenomas and variable expression of PKC beta and gamma in some tumors. When the calcium-independent PKC isozymes (delta, epsilon, and zeta) were localized by in situ hybridization, normal and neoplastic pituitaries expressed abundant mRNA for PKC epsilon, whereas some tumors and one normal pituitary had a few cells positive for PKC zeta mRNA as evaluated by grain density and the number of cells labeled. These results indicate that there is a variable distribution of PKC mRNA isozymes in human pituitary adenomas and that normal pituitaries and pituitary adenoma cells express the mRNA for both the calcium-dependent and some of the calcium-independent PKC isozymes. Chronic treatment with the hypothalamic gonadotropin hormone-releasing hormone and GH-releasing hormone, which increased LH/FSH and GH secretion, respectively, did not increase PKC activity in cultured adenoma cells. The presence of calcium-dependent and calcium-independent PKC isozymes in normal and neoplastic pituitary cells indicates that PKC probably plays a major role in signal transduction in the human pituitary adenomas examined in this study.     

Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas.

Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin-positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis.     

Analysis of epidermal growth factor receptor and activated epidermal growth factor receptor expression in pituitary adenomas and carcinomas.

Epidermal growth factor receptor plays an important role in the pathogenesis of many malignancies. Various growth factors, including epidermal growth factor receptor, have been shown to influence pituitary tumor growth and differentiation. To analyze the role of epidermal growth factor receptor in pituitary tumor development, we examined normal pituitaries (n=8), pituitary adenomas (n=158), and pituitary carcinomas (n=7) for expression of epidermal growth factor receptor protein and messenger RNA using tissue microarrays and RT-PCR. We also examined (a) the expression of phospho-epidermal growth factor receptor, the activated form of epidermal growth factor receptor, in pituitary tumors and normal pituitaries by immunohistochemistry and (b) the effects on epidermal growth factor receptor expression of treating pituitary cells (HP75 cell line) with epidermal growth factor. Epidermal growth factor receptor and the phosphorylated variant expression were present in normal pituitary cells. Epidermal growth factor receptor messenger RNA was also detected in normal pituitaries, pituitary adenomas, and carcinomas by in situ hybridization and RT-PCR. Most pituitary adenomas showed expression of epidermal growth factor receptor and the phosphorylated variant. Nonfunctional adenomas showed higher levels of expression of epidermal growth factor receptor (76 vs 34%) and of phospho-epidermal growth factor receptor (26 vs 8%) as compared to functional adenomas. Five of seven pituitary carcinomas showed strong expression of both epidermal growth factor receptor and phospho-epidermal growth factor receptor. When a human pituitary cell line (HP75) was cultured in the presence of epidermal growth factor receptor, there was an increase in the levels of both epidermal growth factor receptor and phospho-epidermal growth factor receptor after 5 h of treatment, thus confirming that epidermal growth factor receptor signaling was active in pituitary tumors. These results indicate that activated epidermal growth factor receptor is expressed in pituitary adenomas and carcinomas. Higher levels in pituitary carcinomas suggest a role in pituitary tumor progression.     

Immunohistochemical demonstration of cytokeratins in endocrine cells of the human pituitary gland and in pituitary,adenomas

Summary Ten non-neoplastic pituitary glands and 22 pituitary adenomas producing different hormones were studied by immunofluorescence microscopy as well as peroxidase-antiperoxidase and biotin-avidin techniques on frozen sections and formalin-fixed, paraffin-embedded material using antibodies to cytokeratin, vimentin, GFAP, neurofilament protein and different pituitary hormones. The endocrine cells in non-neoplastic pituitary glands as well as in most pituitary adenomas were cytokeratin-positive. The cytoplasmic cytokeratin distribution patterns of non-neoplastic and tumor cells were similar and typical of the type of hormone produced: GH-producing normal cells showed a paranuclear condensation of cytokeratin-reactive intermediate filaments; this accumulation was even further accentuated in GH-producing adenomas resulting in fibrous bodies (Kovacs and Horvath 1978) decorated by cytokeratin antibodies. Prolactin-producing cells showed a less intense cytoplasmic cytokeratin-specific staining with focal paranuclear accentuation in non-neoplastic as well as in neoplastic glands. ACTH-producing cells in normal pituitary glands as well as in adenomas exhibited a strong and more uniform cytoplasmic cytokeratin staining. The cytokeratin reactivity in glycoprotein hormone-producing cells of non-neoplastic tissue and adenomas was weak. Vimentin and GFAP reactivity was confined to agranular folliculo-stellate cells. The specific and different distribution patterns of cytokeratins in pituitary cells can, therefore, provide an (indirect) indication to the production of a specific hormone if immunocytochemistry fails to demonstrate hormone production.Dedicated to Prof. Dr. J.H. Holzner on the occasion of his birthday     

Inactivation of the p16 gene in human pituitary nonfunctioning tumors by hypermethylation is more common in null cell adenomas

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A/MTS1 (p16) gene is associated with loss of expression of p16 protein in pituitary tumors. We analyzed a series of 21 pituitary adenomas and three normal pituitaries along with a human pituitary cell line (HP75) for methylation of exon 1 by methylation-specific PCR, immunohistochemistry, and Western blotting. PCR analysis showed that 5/7 (71%) of null cell adenomas, but only 2/7 (29%) gonadotroph tumors were hypermethylated. In addition, 1 of 2 ACTH tumors but no GH (n=4) or PRL (n=1) adenoma examined were hypermethylated. Immunostaining and Western blot analysis of protein expression supported the methylation-specific PCR analyses. These results show that p16 gene silencing by hypermethylation is more common in null cell adenomas compared to other nonfunctioning adenomas such as gonadotroph tumors and that the role of p16 in the pathogenesis of pituitary adenomas is restricted to specific tumor subtypes. Supported in part by NIH CA90249 and by a grant from the Jarislowsky Foundation.     

Vasoactive intestinal peptide in the human pituitary gland and adenomas. An immunocytochemical study

Recent evidence indicates that vasoactive intestinal peptide (VIP) may be involved in normal pituitary function. Immunocytochemistry was used to localize VIP in human biopsied pituitary adenomas and postmortem anterior pituitary glands. Paraffin sections were immunostained for VIP with the avidin-biotin-peroxidase technique. Strong VIP-like immunoreactivity (VIP-LI) was observed in 16 of 17 prolactinomas, 12 of 14 growth hormone-secreting tumors associated with acromegaly, four of 12 ACTH-secreting tumors, and 14 of 18 nonfunctioning pituitary adenomas. In most cases, VIP was colocalized with the classical pituitary hormones. Six of the 18 nonfunctioning tumors had no demonstrable hormone immunoreactivity; five of these stained strongly for VIP, whereas one was negative. Of 18 normal anterior pituitaries, 12 showed strong diffuse staining for VIP throughout the gland. One pituitary with VIP-LI came from an individual who had undergone pituitary stalk transection. Double-immunoenzyme labeling and immunoelectron microscopy demonstrated VIP-LI in many lactotrophs, scattered thyrotrophs, corticotrophs, and in an occasional gonadotroph. These results suggest the following: 1) VIP is present in more than one cell type in normal and adenomatous human pituitaries; and 2) VIP may be involved in the function and development of pituitary tumors.     

Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas.     

Localization of Epidermal Growth Factor (EGF) and Epidermal Growth Factor Receptor (EGFr) in Human Pituitary Adenomas and Nontumorous Pituitaries: An Immunocytochemical Study

Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFr) were investigated by immunocytochemistry (ICH) in 57 human pituitary adenomas and 10 nontumorous autopsy pituitaries. EGF immunoreactivity was demonstrated in 24 adenomas (42%), representing 23 functioning tumors and 1 nonfunctioning tumor of oncocytic type, and in all nontumorous pituitaries. Among 40 tumors, EGFr was found positive in 15 functioning adenomas (37.5%), representing 50% of them. The presence of both EGF and EGFr was found mainly in corticotroph adenomas (60%) and less frequently in somatotroph and lactotroph adenomas (20%). ICH on serial sections with EGF or EGFr and adrenocorticotrophic hormone (ACTH) or S-100 protein revealed that EGF and EGFr are localized specifically in corticotrophs and EGFr in stellate cells of nontumorous adenohypophysis. These results confirm the presence of EGF and EGFr in human pituitary adenomas and nontumorous pituitaries and highlight their frequent occurrence in hormone-producing adenomas. Further work is required to explore the possibility that EGF and EGFr play a role in hormone production, release, and tumor progression.     

Silencing of the imprinted DLK1-MEG3 locus in human clinically nonfunctioning pituitary adenomas

DLK1-MEG3 is an imprinted locus consisting of multiple maternally expressed noncoding RNA genes and paternally expressed protein-coding genes. The expression of maternally expressed gene 3 (MEG3) is selectively lost in clinically nonfunctioning adenomas (NFAs) of gonadotroph origin; however, expression status of other genes at this locus in human pituitary adenomas has not previously been reported. Using quantitative real-time RT-PCR, we evaluated expression of 24 genes from the DLK1-MEG3 locus in 44 human pituitary adenomas (25 NFAs, 7 ACTH-secreting, 7 GH-secreting, and 5 PRL-secreting adenomas) and 10 normal pituitaries. The effects on cell proliferation of five miRNAs whose expression was lost in NFAs were investigated by flow cytometry analysis. We found that 18 genes, including 13 miRNAs at the DLK1-MEG3 locus, were significantly down-regulated in human NFAs. In ACTH-secreting and PRL-secreting adenomas, 12 and 7 genes were significantly down-regulated, respectively; no genes were significantly down-regulated in GH-secreting tumors. One of the five miRNAs tested induced cell cycle arrest at the G2/M phase in PDFS cells derived from a human NFA. Our data indicate that the DLK1-MEG3 locus is silenced in NFAs. The growth suppression by miRNAs in PDFS cells is consistent with the hypothesis that the DLK1-MEG3 locus plays a tumor suppressor role in human NFAs.