Types Of Human Pituitary Adenomas Research Articles

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.

HESX1 expression in human normal pituitaries and pituitary adenomas

Hesx1 is a paired-like homeobox gene first expressed during mouse embryogenesis in the anterior midline visceral endoderm. As gastrulation proceeds, Hesx1 is expressed in the ventral prosencephalon and, subsequently, at E9.0 appears in the ventral diencephalon and in the thickened layer of oral ectoderm that give rise to Rathke's pouch, the primordium of the anterior pituitary gland. Hesx1 continues to be expressed in the developing anterior pituitary until E11.5 when its transcripts disappear in a spatiotemporal sequence corresponding to progressive pituitary cell differentiation, becoming undetectable by E15.5. In the present study, we investigated whether HESX1 is expressed during adult life in human normal pituitaries and in different types of human pituitary adenomas. We analysed, using quantitative RT-PCR method, three normal pituitaries, seven GH-, two TSH-, two PRL-, one ACTH-secreting adenomas, and seven nonfunctioning pituitary tumors. HESX1 mRNA was found to be expressed in normal pituitaries and in all the pituitary tumors that we have analysed. These results suggest that in humans HESX1 is not turned-off during the adult life as it occurs in mice. Thus, HESX1 in humans might play a role in the maintenance of the anterior pituitary cell types and function, as well as in the differentiation of pituitary adenomas, whose pathogenetic mechanisms remain to be further investigated. This is the first study on HESX1 expression in humans during adult life.

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

Somatostatin receptors in pituitary function, diagnosis and therapy.

Recent studies have demonstrated that human pituitary adenomas express multiple somatostatin receptor (sst) subtypes. The expression of sst subtypes in human pituitary adenomas is highly variable. This variability in sst subtype expression may explain the variable responsiveness of patients with pituitary adenomas to medical treatment with the sst2-preferring SS-analogs octreotide and lanreotide. In human GH-secreting pituitary adenomas, both sst2 and sst5 are involved in the regulation of GH secretion. In prolactinomas, sst5 receptors are the key receptors in regulating responsiveness to SS. The low abundance of sst2 in prolactinomas explains the lack of efficacy of octreotide in lowering the elevated PRL levels in prolactinoma patients. Octreotide and lanreotide successfully suppress TSH levels, including normalization of thyroid hormone levels in the large majority of patients with TSH-secreting pituitary adenomas, probably due to the high level of sst2, expression in the adenomas. Although sst2 receptors are expressed by a significant proportion of gonadotroph and clinically non-functioning pituitary adenomas, the overall efficacy of sst2-preferring SS-analogs seems low in this type of patients. Finally, corticotroph adenomas may express multiple sst subtypes as well. Octapeptide SS-analogs do not lower circulating ACTH levels in patients with untreated pituitary-dependent Cushing's disease, whereas SS and the SS-analog octreotide suppress pathological ACTH release in some patients with Nelson's syndrome. This review discusses the expression and potential role of sst subtypes in the different types of human pituitary adenomas.

Differential regulation of proopiomelanocortin and pituitary-restricted transcription factor (TPIT), a new marker of normal and adenomatous human corticotrophs.

Since the identification of the pituitary-restricted transcription factor Tpit, a novel T-box factor that is only present in mouse in the two pituitary proopiomelanocortin (POMC)-expressing lineages, no information was available on its pattern of expression in human pituitary. We investigated by immunohistochemistry and in situ hybridization the expression of TPIT in normal human anterior pituitary tissue and in several types of human pituitary adenomas (n = 52). TPIT expression was restricted to the nucleus of normal or adenomatous human corticotroph cells. No specific TPIT immunostaining was detectable in all prolactin (PRL)-, GH-, or gonadotropin-secreting adenomas. In situ hybridization studies demonstrated that TPIT transcripts were coexpressed with POMC mRNA in both secreting and silent corticotroph adenomas, and in normal corticotrophs, whereas TPIT mRNA was not detectable in other types of pituitary adenomas. Unlike POMC, TPIT was not up-regulated by adrenalectomy in rats and did not seem down-regulated in the normal pituitary adjacent to human corticotroph microadenomas. TPIT is the only currently known transcription factor selectively expressed in human normal and adenomatous corticotrophs. In human and experimental models, TPIT and its target gene POMC were thus differentially regulated by glucocorticoids. Moreover, TPIT represents a new marker of POMC-expressing pituitary cells.

Expression of Neuro D1 in Human Normal Pituitaries and Pituitary Adenomas

Neuro D1 is a basic helix-loop-helix transcription factor expressed in the endocrine cells of pancreas and in a subset of neurons as they undergo terminal differentiation. In the adult pituitary gland, Neuro D1 is expressed in corticotroph cells and contributes to the corticotroph-specific pro-opiomelanocortin (POMC) transcription by interacting with Pituitary homeobox 1 (Ptx 1) transcription factor. In the present study, we investigated the expression of Neuro D1 in human normal pituitaries and different types of human pituitary adenomas using the RT-PCR and immunohistochemical techniques. Using RT-PCR, Neuro D1 mRNA was found to be expressed in ACTH-secreting adenomas (n = 3) and 6 of 8 non-functioning adenomas. On the other hand, GH-secreting adenomas (n = 5) and PRL-secreting adenomas (n = 3) were completely negative for Neuro D1 mRNA. Immunohistochemically, Neuro D1 was expressed in all ACTH-secreting adenomas (n = 10), and in 14 of 20 nonfunctioning adenomas. In contrast, 3 of 10 PRL-secreting adenomas and 2 of 10 GH-secreting adenomas showed positive Neuro D1 staining in the nuclei. The above results suggest that Neuro D1 contribute to the functional expression and the differentiation of ACTH-secreting adenomas. It also appears from our study that Neuro D1 might play a role in the differentiation of non-functioning adenomas, the mechanism of which remains to be further investigated. This is the first study on Neuro D1 in case of human pituitary adenomas.

Expression of leukemia inhibitory factor in human pituitary adenomas: a morphologic and immunocytochemical study.

Leukemia inhibitory factor (LIF) is a pleiotropic, neuropoietic cytokine found in bovine, murine, and human fetal and adult pituitary cells, mostly in corticotrophs and somatotrophs. In addition, it has been found in a few GH- and ACTH-producing human pituitary adenomas. The aim of our study was to investigate the presence of LIF in various morphologic types of human pituitary adenomas. Ninety-eight operated pituitary adenomas diagnosed by light microscopy and classified by pituitary hormone immunocytochemistry and electron microscopy were studied. Sixty-eight tumors were functioning and included 15 densely granulated (DG) and 10 sparsely granulated (SG) somatotroph (SM) adenomas, 5 lactotroph (LT), 7 mixed SM-LT and 31 corticotroph (CRT) adenomas. The remaining 30 nonfunctioning tumors included 11 gonadotroph (GON) and 19 null cell (NULL) adenomas. For immunocytochemical demonstration of LIF, a specific polyclonal antiserum was applied on formalin-fixed, paraffin-embedded tissues. The immunohistoscore ranging from 1 to 64 grades was determined by multiplying the immunostaining grade (1-4) by the staining intensity grade (1-4), and by the heterogeneity grade (1-4). Ninety adenomas (92%) were variably immunopositive for LIF. LIF was expressed in 77.5% of CRT, 81.8% of GON, 93.3% of DG-SM, and in all SG-SM, LT, SM-LT and NULL adenomas. LT adenomas showed the highest immunostaining grade, followed by SG-SM, GON, NULL, SM-LT, DG-SM, LT and CRT adenomas. GON adenomas showed the highest immunohistoscore, followed by NULL, DG-SM, SM-LT, SG-SM, LT and CRT adenomas. Nonfunctioning tumors showed a significantly higher immunohistoscore compared to functioning adenomas (p < 0.01). We conclude that LIF expression is frequent in all types of functioning and nonfunctioning pituitary adenomas.

High levels of matrix metalloproteinases regulate proliferation and hormone secretion in pituitary cells.

Beside the digestion of the extracellular matrix during tumor invasion and metastasis, more recently, new functions for matrix metalloproteinases (MMPs) have been proposed. We studied the expression and function of these enzymes in pituitary cells. We observed the activities of MMP-2 and MMP-9 together with expression of membrane-type MMP and tissue inhibitor of metalloproteinase-1 in all types of human pituitary adenomas. We found surprisingly high levels of MMP activity and low levels of tissue inhibitor of metalloproteinases, indicating a high level of extracellular matrix-degrading activity in pituitary adenomas. To examine the function of metalloproteinase activity in pituitary cells we used the synthetic MMP inhibitor batimastat. These studies demonstrate that MMPs secreted by pituitary cells can release growth factors anchored to the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. These results define a new additional mechanism for the control of pituitary hormone secretion and indicate new potential therapeutic targets for pituitary adenomas.

Expression of the members of the Ptx family of transcription factors in human pituitary adenomas.

A number of putative transcription factors described in the pituitary have been implicated as key elements in the processes that direct pituitary development. Three recently described proteins, Ptx1, Ptx2, and Ptx3, define a new family of transcription factors, the Ptx subfamily, within the paired-like class of homeodomain factors. In mice, Ptx1 and Ptx2 gene expression has been detected in the area of the pituitary primordium and is maintained throughout development in Rathke pouch and adult pituitary. In the present study, the expression of the Ptx1, Ptx2, and Ptx3 genes was characterized in the normal human pituitary and in the different types of human pituitary adenomas. Although no Ptx3 gene expression could be detected in these tissues, Ptx1 presented with a quite ubiquitous pattern of distribution, being expressed at quite constant levels in normal tissues and in all 60 pituitary tumors analyzed. The pattern of expression of the Ptx2 gene among the different subsets of pituitary adenomas was even more varied. No Ptx2 expression could be detected in corticotroph tumors. In contrast, high levels of Ptx2 messenger ribonucleic acid were measured in the gonadotroph tumors, although no specific correlation to other markers of the gonadotroph lineage differentiation, such as alphaGsu, LHbeta, or FSHbeta, could be evidenced. Finally, Ptx2 was also expressed in pure lactotroph adenomas and not in somatotroph adenomas. Ptx2 is, therefore, the first paired homeodomain pituitary transcription factor differentially expressed in these two lineages, which derive from a common precursor. These results support a role for Ptx2 in the terminal differentiation of somatotroph and lactotroph cell phenotypes.

Growth hormone-releasing hormone receptor (GHRH-R) mRNA expression in human pituitary adenomas: A study by catalyzed reporter deposition-In situ hybridization (CARD-ISH)

Growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene transcription, synthesis, and secretion of growth hormone via growth hormone-releasing hormone receptor (GHRH-R). In a previous study using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blotting, GHRH-R mRNA was detected in all types of pituitary adenomas. On the other hand, in a recentin situ hybridization (ISH) study of 16 adenomas, including GH (n=8), PRL (n=1), ACTH (n=2), gonadotroph (n=4), and null-cell (n=1) adenomas, GHRH-R mRNA expression was observed only in GH adenomas and one gonadotroph adenoma. Thus, the extent of GHRH-R mRNA in pituitary adenomas is not clear. To clarify these different findings, we investigated the expression of GHRH-R mRNA in various types of human pituitary adenomas, including GH (n=7), PRL (n=4), ACTH (n=3), gonadotroph (n=8), and null-cell adenomas (n=13) using conventional ISH and the CARD-ISH technique, which is more sensitive than previous nonisotopic ISH techniques. By conventional ISH, GHRH-R was found in 71% of GH, 50% of PRL, 67% of ACTH, 12% of gonadotroph, and 8% of null-cell adenomas. By catalyzed reporter deposition-in situ hybridization (CARD-ISH), GHRH-R mRNA was expressed in 100% of GH, 50% of PRL, 67% of ACTH, 62% of gonadotroph, and 70% of null-cell adenomas. Hybridization with the control sense probe was consistently negative. These results show that GHRH-R mRNA is expressed in all types of adenomas with GH adenomas showing the strongest signal for GHRH-R mRNA, suggesting that GHRH-R has a role mainly in the function of GH adenomas, but may also play a role in tumor growth and/or function of other types of adenomas. These experiments also show that CARD-ISH can be used to detect low copy numbers of specific mRNAs because of the increased sensitivity of this technique.

Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness.

Epidermal growth factor (EGF) has been localized in several human neoplasms and has been shown to have a significant correlation to prognosis. We investigated the potential role of the EGF receptor (EGF-R) system in pituitary tumorigenesis by examining the expression of EGF and EGF-R in the different types of human pituitary adenomas. EGF was identified by immunohistochemistry in all cell types of the nontumorous adenohypophysis and in all types of morphologically characterized functional (n = 9) and nonfunctional (n = 17) adenomas. To confirm local EGF synthesis, ribonucleic acid (RNA) from human pituitary adenomas was reverse transcribed and PCR amplified. Transcript signals of the expected size were identified, with marked variation in 41 of 48 adenomas. To assess possible secretion in vitro, EGF was measured in pituitary tumor culture medium. No measurable quantities of EGF were present in conditioned culture medium from all 35 adenomas examined, consistent with the rapid uptake of EGF by unoccupied functional EGF-R sites. Using a specific monoclonal antibody that recognizes the extracellular ligand-binding domain of the human EGF-R, we found EGF-R in cells in the normal pituitary and in some functional and nonfunctional adenomas with extremely variable intensity. By RT-PCR, EGF-R messenger RNA (mRNA) expression was also identified, with marked variation in all 48 adenomas examined and in the nontumorous pituitary. The highest degrees of EGF-R mRNA expression were present in somatotroph adenomas and the aggressive silent subtype 3 adenomas. Tumors from patients with recurrent acromegaly demonstrated significantly higher levels of EGF-R mRNA than those from patients with nonrecurrent disease. In conclusion, EGF and EGF-R are expressed in a variable manner in all types of human pituitary adenomas. The overexpression of EGF-R in recurrent somatotroph adenomas and aggressive silent subtype 3 adenomas suggests a selective mechanism for the EGF/EGF-R family in the growth of aggressive pituitary tumors.

In situ hybridization analysis of Pit-1 mRNA and hormonal production in human pituitary adenomas.

The pituitary-specific transcriptional factor, Pit-1, is a member of the POU-domain family which has a role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Recent investigations have suggested the involvement of specific regulation of Pit-1 transcripts in human pituitary adenomas. In this study, we analyzed the expression of Pit-1 gene and Pit-1 product in various human pituitary adenomas using in situ hybridization (ISH) and immunohistochemistry (IHC). Northern hybridization analysis revealed 2.4- and 4.1-kb Pit-1 transcripts in normal pituitary, growth hormone (GH)-, prolactin (PRL)- and thyrotropin (TSH)-secreting adenomas. By ISH analysis, Pit-1 mRNA was detected in 42 (84%) of 50 adenomas. The highest incidence was observed in 15 GH-secreting adenomas and 8 TSH-secreting adenomas, in which Pit-1 mRNA was detected in all cases. Pit-1 mRNA expression was detected in 11 (85%) of 13 PRL-secreting adenomas. In 12 clinically non-functioning adenomas, Pit-1 mRNA was also present in 8 cases, and 5 of these were associated with immunohistochemical expression of Pit-1 product. By combined ISH and IHC, Pit-1 mRNA was often colocalized with GH, PRL or TSH beta immunoreactivities and sometimes colocalized with alpha-subunit of glycoprotein (alpha SU) immunoreactivity. The expression of Pit-1 mRNA in various cell types of human pituitary adenomas in addition to GH, TSH beta and PRL immunoreactivities suggests that Pit-1 may play a role in functional development of pituitary adenomas, including clinically non-functioning adenomas. However, some additional transcriptional factors or enhancers may be required.

Receptors for pituitary adenylate cyclase activating peptides in human pituitary adenomas.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors coupled to adenylate cyclase was investigated in four types of human pituitary adenomas: three null adenomas and five gonadotropin-, three ACTH-, four GH-, and four PRL-producing adenomas. In all samples, except in prolactinomas, PACAP(1-27) and PACAP(1-38) stimulated adenylate cyclase activity equally well and potently (K(act) around 3 nmol). Vasoactive intestinal polypeptide (VIP) was systematically 100- to 300-fold less potent than both PACAPs. In prolactinomas, PACAP(1-27), PACAP(1-38), and VIP were inactive despite a response of the enzyme to guanosine 5'-triphosphate, Gpp(NH)p, forskolin, and fluoride. [125I-AcHis1]PACAP(1-27) binding was detected in all samples except in prolactinomas. In addition, a detailed analysis of receptors was feasible in all five gonadotropin- and in two ACTH-producing adenomas, confirming the existence of selective PACAP receptors that recognized PACAP(1-27) and PACAP(1-38) with similar high affinity (IC50 0.8-1.5 nmol) and VIP with a low affinity (IC50 100 nmol/L).

Human pituitary tumours: studies on genes expression.

Searching for differences in gene expression between different types of human pituitary adenomas, we evaluated the concentration of mRNA from hormonal genes (prolactin and growth hormone) and oncogenes (c-myc and c-fos) in 12 growth hormone-secreting 7 prolactin-secreting and 11 nonsecreting adenomas. We found a positive correlation between clinical diagnoses and hormonal gene expression in all the cases. Our reports indicate the presence of c-myc and c-fos mRNA in all the adenomas evaluated. The concentration of c-myc mRNA was higher in somatotrophic adenomas than in prolactinomas and nonsecreting adenomas whereas c-fos mRNA concentration was similar in the different types of tumours analysed. Oncogenes products, in turn, might stimulate DNA synthesis and cell proliferation and eventually lead to the formation of pituitary adenomas. This is a working hypothesis.

In vitro preservation of human pituitary tumours in organotypic differentiation.

Three types of human pituitary adenomas--growth hormone producing, prolactin producing, as well as endocrinologically inactive ('chromophobe') tumours--were explanted to an in vitro system for organ culture. After one month, surviving hypophyseal cells demonstrated preserved hormone activity and no dedifferentiation of cell characteristics. During the first weeks in culture, close similarity existed between in vivo and in vitro conditions with regard to cell survival/cell morphology and physiological function/hormone release.