Human Anterior Pituitary Research Articles

Morphometric analysis of somatotropic and folliculostellate cells of human anterior pituitary during ageing

Introduction/Objective. In this study, we have pointed out the immunohistomorphometric characteristics of somatotropic (GH) and folliculostellate (FS) cells of the male pituitary gland during ageing. Methods. On histological sections of the pituitary gland of 14 male cadavers of different ages, the GH and FS cells were immunohistochemically labeled with corresponding antibodies, monoclonal anti-GH antibody, and polyclonal anti-S100 antibody, respectively. Immunopositive GH- and FS-cells were further morphometrically analyzed using ImageJ software. Results. The obtained results of morphometric analysis showed that the surface area of GH cells increased significantly with age. In these cells, the nuclear?cytoplasmic ratio gradually decreased and became significantly higher after the age of 70 years. The volume density of GH cells has not changed during ageing, while in FS cells this parameter significantly increased in the cases older than 70 years. The nuclear?cytoplasmic ratio of GH cells is negatively correlated with the volume density of FS cells. Conclusion. Based on the obtained results, we concluded that hypertrophy of GH and FS cells occurs in men with ageing and that correlation between the morphometric parameters of these two cell types indicates their mutual interaction.

Aplicación de la Hematoxilina férrica de M. Heidenhain para el estudio de cortes semifinos (adenohipófisis humana)

Heidenhain´s iron hematoxylin has been one of the most useful methods of classical cytology. Its ability to stain simultaneously many cell and extracellular structures makes it a general purpose histological technique. This current work describes its application to semithin sections, using human adenohypophysis, discussing the staining mechanism and showing his usefulness to evidence cytological variability and to perform image analysis.

Human anterior pituitary's ACTH cells during the aging process: immunohistochemic and morphometric study.

Corticotrophs produce a hormone that stimulates the adrenal gland cortex to secrete glucocorticoids, which in turn have effects on carbohydrate and protein metabolism. Quantification, morphological characteristics, and distribution of corticotrophs in the anterior pituitary and changes in the number and shape of the cells during aging have been examined using immunohistochemical and morphometric methods. The material consisted of 14 anterior pituitaries taken from cadavers at routine autopsy. The tissue was processed by standard histological procedure and the obtained slices were stained by the monoclonal anti-ACTH antibody for corticotrophs identification. Digital images of stained histological sections were analyzed using the morphometric method with the Image J system. The volume density of ACTH positive cells was determined. The cases were classified into three age groups. One-way ANOVA showed that the volume density of the corticotrophs was significantly higher in the second and third group in relation to the first group. The difference in the volume densities of the corticotrophs between the genders was not significant. Morphometric and statistical analyses demonstrated a significant positive correlation between the corticotrophs volume densities and the age of the evaluated cases. Linear regression showed that age significantly predicts corticotrophs volume density. Corticotrophs significantly increase during the life span.

Proteomic Analysis of the Human Anterior Pituitary Gland.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Identification of long non-coding RNAs in the immature and mature rat anterior pituitary

Many long non-coding RNAs (lncRNAs) have been identified in several types of human pituitary adenomas and normal anterior pituitary, some of which are involved in the pathogenesis of pituitary adenomas. However, a systematic analysis of lncRNAs expressed at different developmental stages of normal pituitary, particularly in rats, has not been performed. Therefore, we contrasted two cDNA libraries of immature (D15) and mature (D120) anterior pituitary in rat that were sequenced on an Illumina HiSeq Xten platform, and a total of 29,568,806,352 clean reads were identified. Notably, 7039 lncRNA transcripts corresponded to 4442 lncRNA genes, and 1181 lncRNA transcripts were significantly differentially expressed in D15 and D120. In addition, 6839 protein-coding genes (<100 kb upstream and downstream) were the nearest neighbors of 4074 lncRNA genes. An interaction network of lncRNAs and the follicle-stimulating hormone beta-subunit (FSHb) gene was constructed using the lncRNATargets platform, and three novel lncRNAs were obtained. Furthermore, we detected the expression of the novel lncRNAs and ten highly expressed lncRNAs that were randomly selected through quantitative PCR (qPCR). The rat anterior pituitary lncRNA content identified in this study provides a more in-depth understanding of the roles of these lncRNAs in hormone and reproduction development and regulation in mammals.

Tissue inhibitors of metalloproteinase-expressing cells in human anterior pituitary and pituitary adenoma.

Extracellular matrix (ECM) is essential in tissue physiology and pathologic conditions such as tumorigenesis. It affects tumor cell behavior, proliferation, and metastasis. Pituitary adenomas differ in their clinical characteristics, including ECM deposition, and we recently reported that the characteristics of collagen-producing cells differed between control human anterior pituitary gland and pituitary adenomas. ECM deposition is not defined solely by production; degradation and maintenance are also important. Tissue inhibitors of metalloproteinases (TIMPs) help maintain ECM by inhibiting degradation caused by matrix metalloproteases. The present study attempted to characterize TIMP-expressing cells in the human anterior pituitary. Specimens of human pituitary adenomas and control pituitary were obtained during surgery, and in situ hybridization for TIMP1, TIMP2, TIMP3, and TIMP4, followed by immunohistochemistry, was used to characterize TIMP-expressing cells. TIMP expression exhibited a distinct pattern in the human anterior pituitary. Azan staining showed that fibrous matrix deposition varied among pituitary adenomas and that the area of fibrosis was associated with the number and number of types of TIMP3-expressing cells. These results suggest that TIMPs are important in the maintenance of ECM in human pituitary and that TIMP expressions are altered in fibrosis associated with pituitary adenoma.

NG2 targets tumorigenic Rb inactivation in Pit1-lineage pituitary cells.

The proteoglycan neuron-glial antigen 2 (NG2) is expressed by oligodendrocyte progenitors, pericytes, and some cancerous cells where it is implicated in tumor development. We examined mice with NG2-driven pRb inactivation. Unexpectedly, NG2-Cre:pRb(flox/flox) mice developed pituitary tumors with high penetrance. Adenohypophysial neoplasms developed initially as multifocal lesions; by 1 year, large tumors showed brain invasion. Immunohistochemistry identified these as Pit1-lineage neoplasms, with variable immunoreactivity for growth hormone, prolactin, thyrotropin, and α-subunit of glycoprotein hormones. Other than modest hyperprolactinemia, circulating hormone levels were not elevated. To determine the role of NG2 in the pituitary, we investigated NG2 expression. Immunoreactivity was identified in anterior and posterior lobes but not in the intermediate lobe of the mouse pituitary; in the adenohypophysis, folliculostellate cells had the strongest NG2 immunoreactivity but showed no proliferation in response to Rb inactivation. Pit1-positive adenohypophysial cells were positive for NG2, but corticotroph and gonadotroph cells were negative. RT-PCR revealed NG2 expression in normal human pituitary and human pituitary tumors; immunohistochemistry localized NG2 in nontumorous human adenohypophysis with strongest positivity in folliculostellate cells, and in tumors of all types except corticotrophs. Functional studies in GH4 mammosomatotrophs showed that NG2 increases prolactin (PRL), reduces growth hormone (GH) expression, and enhances cell adhesion without influencing proliferation. In conclusion, NG2-driven pRb inactivation results in pituitary tumors that mimic endocrinologically inactive Pit1-lineage human pituitary tumors. This model identifies a role for NG2 in pituitary cell-type-specific functions and unmasks a protective role from Rb inactivation in folliculostellate cells; it can be used for further research, including preclinical testing of novel therapies.

Human kallikrein 10 expression in surgically removed human pituitary corticotroph adenomas: an immunohistochemical study.

Human kallikrein 10 (hk10), a secreted serine protease, was reported to function as a tumor suppressor. hK10 immunoexpression has been demonstrated in lactrotrophs and corticotrophs of the nontumorous human adenohypophysis. In the present study, for the first time we report hK10 immunoexpression in various surgically removed corticotroph adenoma subtypes. Specimens were fixed in formalin and embedded in paraffin. Immunostaining was performed using the streptavidin-biotin-peroxidase complex method with an hK10-specific rabbit polyclonal antibody. Results showed that the endocrinologically active adrenocorticotropic hormone (ACTH)-producing pituitary tumors and the silent subtypes were immunopositve for hK10. Intensity of staining varied between the different subtypes. Intensity was lowest in the silent subtypes (silent corticotroph subtypes 1 and 2) compared with nontumorous human adenohypophysial corticotrophs, whereas the endocrinologically active subtypes (ACTH-secreting adenomas, corticotroph carcinomas, Crooke cell adenomas, Crooke cell carcinomas), showed the highest hK10 immunoexpression. Immunopositivity in the nuclei of the ACTH-secreting adenomas and carcinomas, as well as dual cytoplasmic and nuclear localization of hK10 in some of the secreting tumor types was an intriguing finding. Immunoexpression of hK10 in the ACTH-secreting tumors as well as in the Crooke cell tumors was significantly increased when compared with the nonfunctioning tumors and in the corticotrophs of nontumorous pituitaries.

EZH2 is expressed in pituitary adenomas and associated with proliferation

Introduction: Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2) and responsible for epigenetic silencing through trimethylation of lysine 27 of histone H3 (H3K27me3). EZH2 is one of the core epigenetic regulator proteins playing a crucial role in cell fate decision, cell cycle regulation, cell differentiation and cancer initiation. It is expressed in several human tumors and often positively associated with high proliferation rates and a poor clinical outcome. We examined the expression of EZH2 in a large cohort of human anterior pituitary glands and related tumor specimens. Methods: A total of 187 human tissue samples (22 normal anterior pituitary glands, 163 pituitary adenomas and 2 pituitary carcinomas) were analyzed by immunohistochemistry, using antibodies against EZH2 and the proliferation marker Ki-67 (Mib1). The extent of EZH2 and Ki-67 protein expression was calculated separately for each slide (percentage of positive tumor cells by counting a minimum of 1000 cells) and correlated with tumor size, invasiveness, age and gender. For statistical analysis we used SPSS. Results: While in normal anterior pituitary tissues EZH2 was totally absent or just restricted to single cells, the related tumors showed enhanced numbers of immuno-positive cells (p ≤0.0005). Here, expression rates were positively associated with the proliferation factor Ki-67 (r = 0.836, p ≤0.0005) but not with any other clinical feature studied. Conclusion: There is evidence that epigenetic alterations are involved in the pathogenesis and tumor biology (i.e. proliferation processes) of pituitary adenomas. Our premature results may indicate a new potential target for prognostic purpose and therapeutic intervention in human pituitary adenomas.

Localization of the aromatase enzyme expression in the human pituitary gland and its effect on growth hormone, prolactin, and thyroid stimulating hormone axis.

The aim of this study is to evaluate aromatase expression in prolactin (PRL), thyroid stimulating hormone (TSH), and growth hormone (GH) secreting cells. Nontumoral human pituitary specimens were obtained from autopsy samples. Aromatase co-expression was determined by double immunohistochemical staining and assessed using H scores. H scores for GH-aromatase co-expression (GH-aromatase), TSH-aromatase co-expression (TSH-aromatase), and PRL-aromatase co-expression (PRL-aromatase) were 83.1 ± 13.1, 95.6 ± 16.1, and 83.7 ± 14.5, respectively. TSH producing cells exhibited the highest H score for co-expression of aromatase (p < 0.001). There was no gender difference in terms of H scores for aromatase expression and double immunohistochemical staining results (p > 0.05 for all). There was a negative correlation between the H scores for aromatase and PRL-aromatase, GH-aromatase and TSH-aromatase, respectively (r = -0.592, p < 0.001; r = -0.593, p < 0.001; r = -0.650, p < 0.001, respectively). Also, H scores for aromatase co-expression of each hormone were negatively correlated with the H scores for the corresponding hormone (r = -0.503, p < 0.001 for PRL-aromatase and PRL; r = -0.470, p < 0.001 for GH-aromatase, and GH; r = -0.641, p < 0.001 for TSH-aromatase and TSH). H scores for mean aromatase, GH-aromatase, TSH-aromatase were invariant of age (p > 0.05 for all). Age was negatively correlated with PRL-aromatase H score (r = -0.373, p = 0.008). Our study demonstrated significant aromatase co-expression in PRL, GH, and TSH secreting cells of the human anterior pituitary gland. The mutual paracrinal regulation between aromatase and three adenohypophyseal hormones indicates that aromatase may have a regulatory role on the synthesis and secretion of these hormones.

The role of TGF-β/Smad signaling in dopamine agonist-resistant prolactinomas

BackgroundProlactinomas are the most common secretory pituitary adenomas. The first line of treatment involves dopamine agonists (DAs); however, a subset of patients is resistant to such therapy. Recent studies suggest that dopamine can up-regulate TGF-β1 synthesis in rat pituitary lactotrophs whereas estradiol down-regulates TGF-β1. To date, the role of TGF-β/Smad signaling in DAs-resistant prolactinomas has not been explored. MethodsHigh-content screening (HCS) techniques, qRT-PCR, Western blot, immunofluorescence and ELISA, were performed to determine the role of TGF-β/Smad signaling in DAs-resistant prolactinomas. ResultsWe reported a significant down-regulation of TGF-β/Smad signaling cascade in DAs-resistant prolactinomas compared to normal human anterior pituitaries. Following treatment with TGF-β1, the dopamine agonist, bromocriptine, and the estrogen antagonist (ER), fulvestrant in GH3 cells, we found that TGF-β1 and fulvestrant caused significant cytotoxicity in a dose- and time-dependent manner and activated Smad3 was detected following exposure to TGF-β1 and fulvestrant. In addition, treating GH3 cells with fulvestrant increased active TGF-β1 levels and decreased PRL levels in a dose-dependent manner. ConclusionTGF-β/Smad signaling pathway may play an important role in DA-resistant prolactinomas and has the potential to be a viable target for the diagnosis and treatment of prolactinomas, particularly in patients who are resistant to DAs.

Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.

Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

Expression of the long non-coding RNAs MEG3, HOTAIR, and MALAT-1 in non-functioning pituitary adenomas and their relationship to tumor behavior

It is becoming evident that long non-coding RNAs (lncRNAs) participate in diverse biological processes via distinct mechanisms. Many lncRNAs have altered expression and likely to have functional roles in tumorigenesis. Although loss of maternally-expressed gene 3 (MEG3) expression has been detected in non-functioning pituitary adenomas (NFPAs), there are no published reports regarding the association between MEG3 expression and the invasive ability of NFPAs. Moreover, the roles of Hox transcript antisense intergenic RNA (HOTAIR) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) in NFPAs have not been examined. To investigate the role of MEG3, HOTAIR, and MALAT-1 in NFPA development and invasion. MEG3, HOTAIR, MALAT-1 and proliferating cell nuclear antigen (PCNA) were detected in 52 NFPA samples and seven normal human anterior pituitaries using real-time quantitative reverse transcription polymerase chain reaction. MEG3 lncRNA levels gradually decreased whereas HOTAIR lncRNA levels gradually increased from normal anterior pituitaries to non-invasive NFPAs to invasive NFPAs. There was a significant association between MEG3 (P < 0.01) and HOTAIR (P < 0.05) expression and the biological behavior of the tumor. Furthermore, PCNA mRNA levels markedly increased in invasive NFPAs compared to non-invasive ones (P < 0.01). In addition, PCNA mRNA negatively correlated with MEG3 lncRNA levels (P < 0.05). MEG3 and HOTAIR expression may correlate with NFPA development and invasion.

Somatostatin Receptors Type 2 and 5 Expression and Localization During Human Pituitary Development

Somatostatin (SRIF), by acting mainly through sst2 and sst5 receptors, is a potent inhibitor of hormonal secretion by the human anterior pituitary gland. However, the pattern of protein expression of these SRIF receptors remains unknown during pituitary development. To get further insights into the physiological role of SRIF receptors in human development and pituitary function, the present study examined the developmental expression of the sst2 and sst5 receptors in the individual cell types of the anterior human pituitary. Thirteen fetal human pituitaries were investigated between 13 to 38 weeks of gestation (WG) by double-labeling immunofluorescence with antibodies raised against sst2 or sst5 receptors and GH, LH, FSH, TSH, or pro-opiomelanocortin proteins. SRIF immunoreactivity in the hypothalamus and median eminence was investigated at the same developmental ages. Immunoreactivity for the sst2 receptor was evident as early as 13 to 15 WG and onward mainly in TSH-, LH-, and FSH-expressing cells, whereas sst5 immunoreactivity was apparent at the late development stages (35-38 WG). GH-expressing cells mainly expressed sst5 immunoreactivity. SRIF-positive fibers and cells were detected as soon as 13 to 16 WG in the hypothalamus and median eminence and their densities increased with gestational age. The early appearance of hypothalamic SRIF cells and fibers suggests a physiological link between SRIF and its receptors during pituitary development. Whereas sst2 receptors might play a primary role in the differentiation and regulation of TSH, LH, and FSH cells, sst5 receptors appear to be mainly involved in GH regulation from birth onward.

Morphometric analysis of the human anterior pituitary's folliculostellate cells during the aging process

Folliculostellate cells represent non-endocrine cells of the anterior pituitary which influence the function of the endocrine cells via paracrine action. Though there is a lack of literature data on their presence during human aging, the aim of this research was to perform the quantification of anterior pituitary folliculostellate cells by the application of immunohistochemical and morphometric methods. The material for the study consisted of 15 anterior pituitaries taken from cadavers at routine autopsy. Their tissue was processed by standard histological procedure and the obtained histological slices were stained by S100 polyclonal antibody. Digital images of stained histological sections were analyzed by morphometric method with ImageJ system. The volume density of S100 positive cells was measured for each case. Results of morphometric and statistical analysis showed a significantly positive correlation between folliculostellate cell volume density and the age of the evaluated cases. Linear regression additionally showed that the age significantly predicts folliculostellate cells volume density in our sample. Further, all cases were classified into three age groups and One Way ANOVA showed that the volume density of folliculostellate cells was significantly higher only in the third age group in relation to the first and the second group, respectively. Volume densities of the first and the second age groups were not significantly different. So, the results of our study pointed to the conclusion that folliculostellate cells presence generally increases with age, but this increase is significant only in the oldest cases and might represent the modality of successful anterior pituitary aging.

OMEGA‐3 POLYUNSATURATED FATTY ACIDS AFFECT LEPTIN RECEPTOR GENE EXPRESSION IN PITUITARY GH4C1 CELL LINE

ABSTRACTLeptin (Ob) and Ob receptor (Ob‐Rb) genes are expressed in human and rodent anterior pituitary, and an autocrine/paracrine loop in the production and regulation of Ob and Ob‐Rb in this tissue has been suggested. As brain membranes contain large amounts of polyunsaturated fatty acids (PUFAs), and PUFA may affect the levels of leptin as well as the receptor's environment, a possible regulatory role of exogenous PUFA on Ob and Ob‐Rb expression in pituitary cell lines was investigated. GH4C1 cells were incubated with arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids and fatty acid incorporation, viability, and gene expression were measured. The results showed that although fatty acids were incorporated in GH4C1 cells at a minimum level, positively affected cell viability without differences, among the different fatty acids. On the contrary, ARA, EPA and DHA showed different effects on Ob‐Rb gene regulation that was upregulated by EPA, downregulated by DHA and not affected by ARA. The effects of leptin co‐incubation are also reported and discussed.PRACTICAL APPLICATIONSData from this paper consolidate the observations regarding the role of n‐3 polyunsaturated fatty acid in gene expression regulation, and thus they could support the recommendation to enrich diet with fish or fish oil with high PUFA content.

Expression of leptin receptor mRNA and the long form splice variant in human anterior pituitary and pituitary adenoma.

Expression of leptin receptor (OB-R) mRNA was detected in the human anterior pituitary as well as in ACTH-secreting and nonsecreting pituitary adenomas by RT-PCR with primers recognizing all receptor splice variants. Primers specific to the long splice variant of the leptin receptor (OB-Rb), containing the putative intracellular signalling domain, also revealed a strong expression in normal and adenomatous anterior pituitaries. These results indicate that the pituitary is a possible target tissue of leptin action and might be involved in leptin regulation of pituitary hormone secretion.

Immunohistochemical distribution of regulatory peptides in the human fetal adenohypophysis

We have studied here the cellular distribution of several regulatory peptides in hormone-producing cells of the human pituitary during the fetal period. Immunohistochemistry was used to show the expression of several regulatory peptides, namely Angiotensin-II, Neurotensin and Galanin, at successive gestational stages and their co-localization with hormones in the human fetal adenohypophysis. Somatotrophs, gonadotrophs and thyrotrophs were differentiated earliest. At gestational week 9, Angiotensin-II immunoreactivity was co-localized only with growth hormone immunoreactivity in somatotrophs, one of the first hormone-producing cells to differentiate. This co-localization remained until week 37. Neurotensin immunoreactivity was present in gonadotrophs and thyrotrophs in week 23, after FSH and TSH hormone differentiation. Galanin immunoreactivity was present in all hormone-producing cell types except corticotrophs. The different pro-opiomelanocortin-derived peptides were detected at different stages of gestation and adrenocorticotrophic hormone immunoreaction was the last to be detected. Our results show an interesting relationship between regulatory peptides and hormones during human fetal development, which could imply that these peptides play a regulatory role in the development of pituitary function.

P75NTR and TrkC Neurotrophin Receptors Demonstrate a Different Immunoreactivity Profile in Comparison to TrkA and TrkB Receptors in Human Normal Pituitary Gland and Adenomas

Background/Aims: Recent knowledge indicates that neurotrophins play a significant role in neuroendocrine systems through their specific receptors TrkA, TrkB, TrkC and low-affinity p75^NTR receptor. TrkA and TrkB receptors have been previously detected in numerous endocrine cells in human anterior pituitary and adenomas. In the present study, the localization of p75^NTR and TrkC along with TrkA and TrkB receptors was investigated. Methods: Semi-serial paraffin-embedded sections of 5 human normal pituitaries and 30 adenomas were immunostained using specific antibodies. Results: Expression of p75^NTR receptor was demonstrated in the intricate capillary and reticulin network in the anterior pituitary and in the pericapillary tissue and pituicytes in the posterior lobe. p75^NTR immunoreactivity was absent from all adenomas. In normal anterior pituitary, a few scattered cells showed weak TrkC immunoreactivity in contrast to a high percentage of endocrine cells distributed throughout the pars distalis and pars intermedia which exhibited strong TrkA and/or intermediate TrkB immunoreactivity. Double immunohistochemistry demonstrated TrkA immunoreactivity in more than 80% of lactotropes and 70% of corticotropes and to a lesser extent in other cell types. Furthermore, in the majority of adenomas, independently of type, sex and age, a high percentage of TrkA- and/or TrkB-positive cells was detected. Interestingly, TrkC expression appeared to be increased in some adenomas compared to normal pituitary. Endothelial cells and perivascular connective tissue were always TrkB-immunostained. Conclusion: The above findings support a potential role of all neurotrophins, through their different receptors, in pituitary functions.

Involvement of the calcium-binding protein secretagogin in pituitary adenoma hormone production

Calcium is a potent intracellular signal transducer whose fluctuations control numerous cellular processes among them hormone production. Secretagogin (SCGN) is an intracytoplasmatic protein belonging to the EF-hand calcium binding protein superfamily, which was initially isolated from a human pancreatic cDNA library and was shown to be involved in the regulation of insulin synthesis and secretion. Since SCGN was also detected in neuroendocrine cells in the anterior pituitary, we have studied the expression of SCGN both in normal and tumoral pituitary and have started to elucidate its role in pituitary cells. Our preliminary results obtained by Real time PCR analysis showed the expression of SCGN in a group of 4 normal human anterior pituitary glands and in 26 different pituitary adenomas. In the tumours, SCGN mRNA was downregulated in 2/4 somatotrophinomas, 6/7 nonfunctioning pituitary adenomas, 4/6 prolactinomas, 3/6 corticotrophinomas and 2/2 thyrotrophinomas when compared to the expression in normal pituitary tissue. The co-transfection of a rat SCGN expression plasmid with prolactin-luc reporter plasmid, GH-luc plasmid and POMC-luc reporter plasmid, respectively in mammosomatotroph GH3 cells and in corticotroph AtT-20 cells, showed that SCGN overexpression was able to induce a 46% increase in the activity of the prolactin gene promoter and a 60% increase in the activity of the GH gene promoter, while in AtT-20 cells the activity of POMC gene promoter showed an increase of 66%. The analysis of the different promoter elements showed an activation of CRE element in GH3 cells after SCGN plasmid co-transfection and an increase in cAMP production. In AtT-20 cells SCGN plasmid co-transfection was able to induce an increase in the activities of AP-1, Nbr-E and NurrRE elements, which was also accompanied by an increase in cAMP production. These preliminary findings suggest the involvement of SCGN in controlling the synthesis of pituitary hormones, therefore constituting a potential target for inhibiting the hormone production.