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.     

Growth hormone-releasing hormone receptor mRNA in acromegalic pituitary tumors.

The growth hormone (GH)-releasing hormone receptor (GHRH-R) has been recently cloned and found to be a member of a new family of seven transmembrane receptors that includes secretin, vasoactive intestinal peptide, calcitonin, and corticotropin-releasing factor. GHRH-R mRNA has been demonstrated by Northern blot analyses to be present specifically in the anterior pituitary gland. To determine the precise cellular localization of this receptor in normal anterior pituitary and pituitary adenomas, GHRH-R mRNA was analyzed in 2 normal human pituitary glands and 16 human pituitary adenomas using in situ hybridization. GHRH-R was specifically localized in somatotroph cells in the normal pituitary. In the adenomas, all GH-producing adenomas originating from acromegalic patients demonstrated up-regulation of GHRH-R mRNA when compared with levels in the normal pituitary. Only one of five clinically nonfunctioning adenomas, a gonadotroph luteinizing hormone/follicle-stimulating hormone-positive adenoma, exhibited up-regulation of this receptor message. Adrenocorticotrophic hormone-secreting and prolactin-secreting adenomas did not express GHRH-R message. In summary, GHRH-R is specifically expressed in somatotrophs and GH-producing adenomas, suggesting that GHRH-R may influence GH release in adenomas similar to this receptor's actions in the normal somatotrophs and may be involved in the growth of GH-secreting 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.     

Retention of peptide hormones during partial secretion in pituitary somatotrophs and corticotrophs

The secretion of peptide hormones during exocytosis of an individual vesicle can result in either complete discharge of vesicle content or can occur in a partial manner in which some hormone is retained during transient fusion. In anterior pituitary lactotrophs, the retained hormone prolactin was internalized and recycled into a pool of vesicles that underwent preferential use during subsequent exocytic stimulations [Bauer et al., (2004) J Cell Sci. 117:2193–2202]. The aim of the present study was to determine whether retention and preferential recycling of retained hormones occurred in other anterior pituitary cells. Stimulation of somatotrophs with high K⁺ resulted in 50 discrete puncta per cell that were positive for growth hormone immunoreactivity. Identical stimulation of corticotrophs resulted in 150 puncta per cell that were anti-adrenocorticotrophic hormone (ACTH) positive. However, unlike what was observed for lactotrophs, the number of structures containing retained growth hormone and ACTH decreased to less than 10% of the initial value in 80 min in somatotrophs and in less than 10 min in corticotrophs. Our results indicate that functional recycling of retained hormones is not shared by all anterior pituitary cell types.     

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.     

Galanin immunoreactivity in neuroendocrine tumors.

We investigated immunoreactivity for galanin, a 29-amino acid peptide, in formalin-fixed, paraffin-embedded sections of 123 neuroendocrine tumors. Galanin-immunoreactive cells were found in one of 12 hypothalamic gangliocytomas, nine of 18 adrenal pheochromocytomas, nine of 14 pituitary corticotroph adenomas, and one of two thymic endocrine tumors. In pheochromocytomas, galanin-immunoreactive cells were seen either singly or in clusters. In corticotroph adenomas, many tumor cells were positive for galanin, indicating colocalization of corticotropin and galanin in the same tumor cells. No galanin-immunoreactive cells were noted in four extra-adrenal paragangliomas; 10 medullary carcinomas of the thyroid; 35 endocrine tumors arising in the lung, pancreas, and gastrointestinal tract; and 28 pituitary adenomas composed of cells other than corticotrophs. In nontumorous counterparts of these neuroendocrine tumors, galanin immunoreactivity was observed in nerve cells of the hypothalamus, nerve fibers of the duodenum, and adenohypophyseal cells corresponding to corticotrophs. These findings indicate that galanin expression in neuroendocrine tumors is uncommon and restricted to some tumor types.     

Reversible transdifferentiation: interconversion of somatotrophs and lactotrophs in pituitary hyperplasia.

Previous studies conclusively demonstrated transformation of somatotrophs into bihormonal mammosomatotrophs in gestational lactotroph hyperplasia during pregnancy. Similar transdifferentiation of somatotrophs into thyrotrophs through bihormonal intermediate thryrosomatotrophs was documented during thyrotroph hyperplasia in both rodent and human pituitaries in hypothyroidism. The cessation of the stimulation resulted in reversal of the process in both conditions. The conversion of lactotrophs into somatotrophs was suggested but not documented previously in the human gland. The present study was undertaken to investigate cases of somatotroph hyperplasia by transmission electron microscopy, immunoelectron microscopy using double immunogold labeling for growth hormone and prolactin, as well as combined immunocytochemistry and in situ hybridization. Adenohypophysial tissue was removed from a 38-year-old man and a 29-year-old woman with long-standing acromegaly due to ectopic overproduction of growth hormone-releasing hormone (GRH) by bronchial carcinoid tumors. For comparison, two pituitary biopsies were studied: one from a 38-year old woman with idiopathic lactotroph hyperplasia and one from a 14-year-old boy with secondary lactotroph hyperplasia due to a suprasellar craniopharyngioma. In the patients with somatotroph hyperplasia, the prevailing cell type was the hyperplastic somatotroph joined by mammosomatotroph deriving from lactotrophs, whereas monohormonal lactotrophs were rare. The predominance of mammosomatotrophs and active lactotrophs was documented in the patient with idiopathic lactotroph hyperplasia, whereas the case of the patient with secondary lactotroph hyperplasia was characterized by monohormonal lactotrophs and somatotrophs, but mammosomatotrophs were rare. That finding in the pituitary of the boy suggests that participation of mammosomatotrophs in lactotroph hyperplasia is not unconditional Our findings conclusively demonstrate conversion of lactotrophs into mammosomatotrophs during somatotroph hyperplasia, providing further evidence for the potential of reversible transdifferentiation between somatotrophs and lactotrophs in response to functional demand.     

Characterization of pituitary cell populations in rats with induced polycystic ovaries

Several hypotheses have been proposed about the pathogenesis of polycystic ovarian syndrome (PCOS), however, the fundamental physiological interactions that initiate the development of follicular cysts have not yet been elucidated. Hence, in this study the proliferation, density and population of gonadotrophs, mammotrophs, somatotrophs and corticotrophs of the pituitary glands of rats with induced follicular cysts have been investigated by 2 experimental models (continuous light exposition and estradiol valerate-treated rats). Specific immunoreactivity associated with follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone and adrenocorticotropic hormone was evaluated by immunohistochemistry with specific hormone antibodies and proliferation of secretory cells by their colocalization (double-labeling) with proliferating cell nuclear antigen. The results indicate a reduction in the density and proliferation of gonadotrophs in both experimental groups. A reduction in the average density, proliferation and population of lactotrophs and corticotrophs was also observed in estradiol valerate-treated animals. However, no significant differences were found in somatotrophs. The present study contributes to the information about alterations of some cell populations that occur in the pituitary gland of rats with polycystic ovaries, and will enhance our understanding of the pathogenesis of this disease.     

Corticotroph (Basophil) invasion of the pars nervosa in the human pituitary: Localization of proopiomelanocortin peptides,galanin and peptidylglycine α-amidating monooxygenase-like immunoreactivities

Corticotroph (basophil) invasion or the migration of corticotroph cells into the pars nervosa of the human pituitary gland was found in 35 of 767 (4.4%) consecutive pituitaries obtained at autopsy. The degree of invasion increased with patient age and extensive invasion was more common in men than in women. Immunoreactive ACTH, β-MSH, α-MSH, and galanin were detected both in the anterior lobe and invading corticotroph cells in approximately equal frequency. Fewer cells stained positively for α-MSH than for the three other peptides in both the anterior lobe and invading corticotrophs. Twelve corticotropic pituitary adenomas obtained surgically from patients with Cushing’s disease were also examined and expressed varying degrees of immunoreactivity for ACTH, α MSH, β-MSH and galanin. Staining for all major pituitary hormones revealed only ACTH in the invading basophil cells. Peptidylglycine α-amidating monooxygenase (PAM) was present in the anterior pituitary, in invading corticotroph cells, and in some cells lining the cysts of the pars intermedia zone. PAM immunoreactivity was also detected in 4/12 corticotroph adenomas. These results indicate that corticotroph cells invading the pars nervosa are immunohistochemically similar to anterior lobe corticotrophs and have the ability to amidate various peptides such as proopiomelanocortin cleavage products and galanin with PAM.     

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.     

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.     

Pituitary adenomas and granular cell tumors. Incidence, cell type, and location of tumor in 100 pituitary glands at autopsy.

Incidentally detected pituitary adenomas were investigated in 100 pituitary glands at autopsy to determine the number, cell type, and location of tumors, and the presence of coexisting granular cell tumors in the neurohypophysis. Pituitary glands were sagittally sectioned at 1.5-mm intervals in toto and embedded in 1 cassette to orient location of each tumor. Twenty-four pituitary glands harbored adenomas, most smaller than 3 mm and the largest 6 x 5 x 4 mm. Two pituitary glands contained double adenomas of immunocytochemically different cell types. Of the 26 adenomas, 10 had lactotrophs, 2 had mixed lactotrophs-somatotrophs, 1 had mixed lactotrophs-luteinizing hormone cells, and 12 were nonfunctioning. One adenoma with adenocorticotropic hormone cells was also detected. Thus 25 of 26 (96%) adenomas were either lactotrophic or nonfunctioning; this percentage is much higher than that of surgically resected tumors. Twenty-two tumors were contiguous with or adjacent to the capsule from which the adenomas originated. Nine granular cell tumors were noted in the neurohypophysis; 3 coexisted with pituitary adenomas. Fourteen additional cases revealed small granular cell nests. Thus the incidental finding of nonfunctioning pituitary adenomas is relatively common in adults (24% of cases in this study), and the coexistence of pituitary adenomas and granular cell tumors may suggest a possible histogenic connection between anterior and posterior pituitary tumorigenesis.     

Glucocorticoid receptor expression in nontumorous human pituitaries and pituitary adenomas

Glucocorticoids have multiple actions, including a suppressive feedback effect on pituitary corticotrophs via the glucocorticoid receptor (GR). By immunocytochemistry, we studied GR expression in 86 surgically removed various pituitary adenoma types. Ten cases contained nontumorous pituitary fragments, which were suitable for immunocytochemical investigation. In addition, 30 autopsy-obtained pituitaries, 10 of them containing incidental microadenomas, were examined as well. Using a polyclonal GR antibody, the streptavidin-biotin-peroxidase complex method revealed nuclear and/or cytoplasmic GR immunoreactivity in many nontumorous corticotrophs and other adenohypophysial cell types and in S-100 protein immunopositive stellate cells. Cellular localization was confirmed by double immunostaining. Pars intermedia corticotrophs, posterior lobe axons, Herring bodies, and pituicytes as well as several endothelial cells lining the capillaries were also immunopositive. GR immunoreactivity was also demonstrated in many GH, PRL, ACTH, TSH, FSH, LH α-subunit producing adenomas, null cell adenomas, and oncocytomas. The extent and degree of immunostaining varied considerably from case to case. Suppressed corticotrophs showing the Crooke’s hyaline change due to glucocorticoid excess were present in the nontumorous pituitaries of patients with Cushing’s disease and in those treated with pharmacologic doses of glucocorticoids. Many suppressed nontumorous corticotrophs exhibited only weak or no GR immunopositivity, indicating GR downregulation accompanied by cellular injury. Study of autopsy obtained pituitaries for GR yielded inconclusive results indicating that autopsy obtained adenohypophyses are not suitable for the immunocytochemical investigation of GR.     

VEGF and CD31 Association in Pituitary Adenomas

Pituitary tumors are usually less vascularized than the normal pituitary, and the role of angiogenesis in these adenomas is contentious. Appraisal of microvascular density and expression of the potent angiogenic vascular endothelial growth factor (VEGF) by immunohistochemistry has yielded controversial results, as a broad spectrum of immunostaining can be found. We determined the protein expression of VEGF and CD31, an endothelial marker, in a series of 56 surgically removed pituitary adenomas using Western blot assay. Prolactinomas had higher VEGF protein expression compared to nonfunctioning or ACTH- and GH-secreting adenomas, while CD31 was similar in the different adenoma histotypes. VEGF and CD31 were not affected by sex, age, years of adenoma evolution, or proliferation rate (Ki67 and PCNA) for all adenoma types. Only in nonfunctioning adenomas CD31 concentration increased significantly with age. There was a positive correlation between CD31 and VEGF expression when all adenoma histotypes were considered, or when prolactinomas and nonfunctioning adenomas were evaluated separately. The positive association of VEGF and CD31 expression suggests the participation of angiogenesis in adenoma development, while epithelial cell proliferation in pituitary tumors is not directly related to VEGF or CD31 expression, and other factors, such as primary genetic alterations may be involved.     

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.