Anterior Pituitary Cells Research Articles

Calcium/calmodulin-dependent kinases can regulate the TSH expression in the rat pituitary.

The endocrine secretion of TSH is a finely orchestrated process controlled by the thyrotropin-releasing hormone (TRH). Its homeostasis and signaling rely on many calcium-binding proteins belonging to the "EF-hand" protein family. The Ca2+/calmodulin (CaM) complex is associated with Ca2+/CaM-dependent kinases (Ca2+/CaMK). We have investigated Ca2+/CaMK expression and regulation in the rat pituitary. The expression of CaMKII and CaMKIV in rat anterior pituitary cells was shown by immunohistochemistry. Cultured anterior pituitary cells were stimulated by TRH in the presence and absence of KN93, the pharmacological inhibitor of CaMKII and CaMKIV. Western blotting was then used to measure the expression of these kinases and of the cAMP response element-binding protein (CREB). TSH production was measured by RIA after time-dependent stimulation with TRH. Cells were infected with a lentiviral construct coding for CaMKIV followed by measurement of CREB phosphorylation and TSH. Our study shows that two CaM kinases, CaMKII and CaMKII, are expressed in rat pituitary cells and their phosphorylation in response to TRH occurs at different time points, with CaMKIV being activated earlier than CaMKII. TRH induces CREB phosphorylation through the activity of both CaMKII and CaMKIV. The activation of CREB increases TSH gene expression. CaMKIV induces CREB phosphorylation while its dominant negative and KN93 exert the opposite effects. Our data indicate that the expression of Ca2+/CaMK in rat anterior pituitary are correlated to the role of CREB in the genetic regulation of TSH, and that TRH stimulation activates CaMKIV, which in turn phosphorylates CREB. This phosphorylation is linked to the production of thyrotropin.

DNA methylation-based signatures classify sporadic pituitary tumors according to clinicopathological features.

Distinct genome-wide methylation patterns cluster pituitary neuroendocrine tumors (PitNETs) into molecular groups associated with specific clinicopathological features. Here we aim to identify, characterize, and validate methylation signatures that objectively classify PitNET into clinicopathological groups. Combining in-house and publicly available data, we conducted an analysis of the methylome profile of a comprehensive cohort of 177 tumors (Panpit cohort) and 20 nontumor specimens from the pituitary gland. We also retrieved methylome data from an independent PitNET cohort (N = 86) to validate our findings. We identified three methylation clusters associated with adenohypophyseal cell lineages and functional status using an unsupervised approach. Differentially methylated probes (DMP) significantly distinguished the Panpit clusters and accurately assigned the samples of the validation cohort to their corresponding lineage and functional subtypes memberships. The DMPs were annotated in regulatory regions enriched with enhancer elements, associated with pathways and genes involved in pituitary cell identity, function, tumorigenesis, and invasiveness. Some DMPs correlated with genes with prognostic and therapeutic values in other intra- or extracranial tumors. We identified and validated methylation signatures, mainly annotated in enhancer regions that distinguished PitNETs by distinct adenohypophyseal cell lineages and functional status. These signatures provide the groundwork to develop an unbiased approach to classifying PitNETs according to the most recent classification recommended by the 2017 WHO and to explore their biological and clinical relevance in these tumors.

Plasticity of Anterior Pituitary Gonadotrope Cells Facilitates the Pre-Ovulatory LH Surge.

Gonadotropes cells located in the anterior pituitary gland are critical for reproductive fitness. A rapid surge in the serum concentration of luteinizing hormone (LH) secreted by anterior pituitary gonadotropes is essential for stimulating ovulation and is thus required for a successful pregnancy. To meet the requirements to mount the LH surge, gonadotrope cells display plasticity at the cellular, molecular and morphological level. First, gonadotrope cells heighten their sensitivity to an increasing frequency of hypothalamic GnRH pulses by dynamically elevating the expression of the GnRH receptor (GnRHR). Following ligand binding, GnRH initiates highly organized intracellular signaling cascades that ultimately promote the synthesis of LH and the trafficking of LH vesicles to the cell periphery. Lastly, gonadotrope cells display morphological plasticity, where there is directed mobilization of cytoskeletal processes towards vascular elements to facilitate rapid LH secretion into peripheral circulation. This mini review discusses the functional and organizational plasticity in gonadotrope cells including changes in sensitivity to GnRH, composition of the GnRHR signaling platform within the plasma membrane, and changes in cellular morphology. Ultimately, multimodal plasticity changes elicited by gonadotropes are critical for the generation of the LH surge, which is required for ovulation.

TGFBR3L—An Uncharacterised Pituitary Specific Membrane Protein Detected in the Gonadotroph Cells in Non-Neoplastic and Tumour Tissue

Simple SummaryPituitary neuroendocrine tumours originate from the endocrine cells of the anterior pituitary gland and may develop from any of the cell lineages responsible for producing the different pituitary hormones. The details related to tumour differentiation and hormone production in these tumours are not fully understood. The aim of our study was to investigate an uncharacterised pituitary enriched protein, transforming growth factor beta-receptor 3 like (TGFBR3L). The TGFBR3L protein is highly expressed in the pituitary compared to other organs. We found the protein to be gonadotroph-specific, i.e., detected in the cells that express follicle-stimulating and luteinizing hormones (FSH/LH). The gonadotroph-specific nature of TGFBR3L, a correlation to both FSH and LH as well as an inverse correlation to membranous E-cadherin and oestrogen receptor β suggests a role in gonadotroph cell development and function and, possibly, tumour progression. Here, we report the investigation of transforming growth factor beta-receptor 3 like (TGFBR3L), an uncharacterised pituitary specific membrane protein, in non-neoplastic anterior pituitary gland and pituitary neuroendocrine tumours. A polyclonal antibody produced within the Human Protein Atlas project (HPA074356) was used for TGFBR3L staining and combined with SF1 and FSH for a 3-plex fluorescent protocol, providing more details about the cell lineage specificity of TGFBR3L expression. A cohort of 230 pituitary neuroendocrine tumours were analysed. In a subgroup of previously characterised gonadotroph tumours, correlation with expression of FSH/LH, E-cadherin, oestrogen (ER) and somatostatin receptors (SSTR) was explored. TGFBR3L showed membranous immunolabeling and was found to be gonadotroph cell lineage-specific, verified by co-expression with SF1 and FSH/LH staining in both tumour and non-neoplastic anterior pituitary tissues. TGFBR3L immunoreactivity was observed in gonadotroph tumours only and demonstrated intra-tumour heterogeneity with a perivascular location. TGFBR3L immunostaining correlated positively to both FSH (R = 0.290) and LH (R = 0.390) immunostaining, and SSTR3 (R = 0.315). TGFBR3L correlated inversely to membranous E-cadherin staining (R = −0.351) and oestrogen receptor β mRNA (R = −0.274). In conclusion, TGFBR3L is a novel pituitary gland specific protein, located in the membrane of gonadotroph cells in non-neoplastic anterior pituitary gland and in a subset of gonadotroph pituitary tumours.

Control of the Anterior Pituitary Cell Lineage Regulator POU1F1 by the Stem Cell Determinant Musashi.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin's mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3' untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

Proposal of a clinically relevant working classification of pituitary neuroendocrine tumors based on pituitary transcription factors

The immunohistochemistry (IHC) characterization of pituitary transcription factors (PTFs) PIT1, TPIT, and SF1, which enable the identification of three different adenohypophyseal cell lines, has been incorporated into the latest classification system of the World Health Organization (WHO) for pituitary adenomas. This change overturns the concept of the adenoma as solely a hormone producer and classifies these tumors based on their cell lineage. The aim of the study was to provide a diagnostic algorithm, based on IHC expression of hypophyseal hormones with potential use in diagnostic practice, contributing to an improved classification of pituitary adenomas. Our sample included 146 pituitary adenomas previously classified based on hormonal subtypes by IHC (former 2004 WHO criteria) and re-evaluated after the IHC quantification of PIT1, TPIT, and SF1 expression, under WHO 2017 recommendations. We assessed the correlation between expression of PTFs and the classification as per hormonal IHCand correlated clinicopathological profiles based on PTFs. The IHC study of PTFs allowed reclassification of 82% of tumors that were negative for all pituitary hormones, with 21 positive cases for SF1 (reclassified as gonadotroph tumors), 1 positive case for TPIT (reclassified as a corticotroph tumor), and 4 positive cases for PIT1. Using SF1 enabled detection of a substantial portion of gonadotroph tumors, reducing the estimated prevalence of null cell tumors to less than 5%, and identification of plurihormonal pituitary neuroendocrine tumors with PIT1-SF1 coexpression and hormone-negative PIT1s, a group in which we did not observe differences in the clinical behavior compared with the rest of the tumors of the same cell lineage.Our results suggest that applying a diagnostic algorithm based on the study of PTFs could contribute to improving the classification of pituitary adenomas. By adding TPIT assessment, we propose a two-step algorithm, with hypophyseal hormones being used in a selective modality, depending on initial results.

Requirement of FAT and DCHS protocadherins during hypothalamic-pituitary development.

Pituitary developmental defects lead to partial or complete hormone deficiency and significant health problems. The majority of cases are sporadic and of unknown cause. We screened 28 patients with pituitary stalk interruption syndrome for mutations in the FAT/DCHS family of protocadherins that have high functional redundancy. We identified 7 variants, 4 of which are putatively damaging, in FAT2 and DCHS2 in 6 patients with pituitary developmental defects recruited through a cohort of patients with mostly ectopic posterior pituitary gland and/or pituitary stalk interruption. All patients had growth hormone deficiency, and 2 presented with multiple hormone deficiencies and small glands. FAT2 and DCHS2 were strongly expressed in the mesenchyme surrounding the normal developing human pituitary. We analyzed Dchs2–/– mouse mutants and identified anterior pituitary hypoplasia and partially penetrant infundibular defects. Overlapping infundibular abnormalities and distinct anterior pituitary morphogenesis defects were observed in Fat4–/– and Dchs1–/– mouse mutants, but all animal models displayed normal commitment to anterior pituitary cell types. Together our data implicate FAT/DCHS protocadherins in normal hypothalamic-pituitary development and identify FAT2 and DCHS2 as candidates underlying pituitary gland developmental defects such as ectopic pituitary gland and/or pituitary stalk interruption.

STAFİLOKOKK İNFEKSİYASI VƏ HİPOFİZAR-ADRENAL-TİROİD SİSTEMİN PATOMORFOLOJİ XARAKTERİSTİKASI

During the staphylococcal infection, changes in the interaction of glandular cells, dystrophic and disorganizing pathologies in tissues, especially acute structural and hemodynamic changes in the stroma of the glands in the pituitary-adrenal-thyroid system, develop from the first day of the experiment. At the end of the experiment, on the background of a decrease in exudative processes, fibroplastic reactions are significantly activated, resulting in signs of incomplete regeneration – mainly sclerotic processes and cystic-atrophic changes in the parenchyma. Structural changes in tissues in the early stages of staphylococcal infection and the dynamics of development are characterized by specific symptoms in each of the glands. Since the pituitary gland is exposed to endogenous and exogenous factors earlier and more often than the adrenal glands, and the adrenal glands are earlier than the thyroid gland, dystrophic and destructive changes in the pituitary and adrenal glands are more pronounced at the early stage of the experiment. These morphological changes can change the hormonal status of the body and lead to dysfunction of the endocrine system as a whole – a decrease in the functional activity of the glands to some extent, and even inhibition of adenohypophyseal cells. Key words: staphylococcal infection, peritonitis, pituitary, adrenal and thyroid glands

Cell-Type-Specific Expression Pattern of Proton-Sensing Receptors and Channels in Pituitary Gland

In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of this study is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G-protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions.

Cytokeratin profiles in pituitary neuroendocrine tumors

The presence and patterns of keratins are critical in the classification of pituitary neuroendocrine tumors. A large body of literature has included information about the staining patterns of pituitary tumors and tissues with the CAM 5.2 antibody. During an antibody validation for clinical use, we carried out staining of a series of 29 surgically resected pituitary cases containing 31 pituitary neuroendocrine tumors that were tested for CAM 5.2 as well as for cytokeratin(CK) 7, 18, 19, and 20 and the pan-keratin cocktail AE1/AE3. The results showed an almost identical staining pattern for CK18 and CAM 5.2; however, CAM 5.2 yielded more intense staining, whereas CK18 provided more delicate results. Staining results using AE1/AE3 were satisfactory but generally less intense; however, this marker was more specific, identifying keratin expression in one tumor that was negative with CAM 5.2. CK19 is expressed in nontumorous adenohypophysis but was less frequently positive in tumors; somatotroph and corticotroph tumors were negative for CK19, but CK19 antibody highlighted follicular cells in some gonadotroph tumors. CK7 and CK20 were negative in all pituitary tissues tested. Our findings underscore the role for CAM 5.2 and CK18 as the most valuable to identify specific alterations in adenohypophysial cells and their tumors; there is also a role for AE1/AE3 to verify the epithelial nature of pituitary neuroendocrine tumors that are negative for CAM 5.2 and CK18.

Structure, Function, and Morphology in the Classification of Pituitary Neuroendocrine Tumors: the Importance of Routine Analysis of Pituitary Transcription Factors.

The traditional approach to the diagnosis of primary adenohypophyseal cell proliferations uses hormone immunohistochemistry to classify pituitary neuroendocrine tumors (PitNETs). The routine application of immunolocalization of pituitary transcription factors (SF1, PIT1, TPIT, ERα, and recently GATA3) along with adenohypophyseal hormones has taught us critical lessons that are discussed in this communication. We point out that appropriate patient care requires accurate diagnosis and is critical in the era of precision medicine. A misdiagnosis can result in far greater health care costs than the cost of accurate tumor classification and may have other unintended consequences. We provide additional insights about confusing findings in genomic studies, emphasizing that high-quality pathology is essential for strong science and translational research.

Quercetin 3-O-malonylglucoside in the leaves of mulberry (Morus alba) is a functional analog of ghrelin.

Mulberry (Morus alba) leaf is traditionally consumed as a functional tea with remedial effects, such as preventing aging-related diseases. Two similar compounds, quercetin 3-O-malonylglucoside, and kaempferol 3-O-malonylglucoside, were detected in mulberry leaves and found to be structural recombinant composites of teaghrelin and emoghrelin, two classes of non-peptidyl compounds functionally identified as analogs of ghrelin. Molecular modeling showed that these two mulberry compounds were able to enter and interact with the ghrelin receptor and theoretical calculation revealed that they were similar to emoghrelin but slightly weaker than teaghrelin in terms of interaction with the receptor. The relatively abundant compound, quercetin 3-O-malonylglucoside was subjected to a bioactivity assay, and the result confirmed that it was able to increase the growth hormone secretion of rat anterior pituitary cells. It seems that quercetin 3-O-malonylglucoside is also a functional analog of ghrelin and presumably a key ingredient for the anti-aging activity of mulberry leaves. PRACTICAL APPLICATIONS: According to this study, quercetin 3-O-malonylglucoside and kaempferol 3-O-malonylglucoside are suggested to serve as active ingredients in tea products prepared from mulberry leaves. Contents of these two compounds might be used as key factors for breeding or screening mulberry varieties for commercial cultivation. Moreover, water extract of mulberry leaves containing these compounds can be used as an adequate supplement for functional food.

Anterior pituitary gland synthesises dopamine from l-3,4-dihydroxyphenylalanine (l-dopa).

Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l-3,4-dihydroxyphenylalanine (l-dopa). Accordingly, we investigated the expression of aromatic l-amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l-dopa and its release in vitro. Then, we explored the effects of l-dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l-dopa. Also, the presence of l-dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l-dopa. In addition, l-dopa reduced corticotrophin-releasing hormone-stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD-1015. Moreover, DA formation from l-dopa increased apoptosis and decreased proliferation. However, in the presence of NSD-1015, l-dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l-dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l-dopa exerts direct actions independently from its metabolisation to DA.

SAT-298 Integrative Single-Cell Transcriptomic and Epigenomic Landscape of Mouse Anterior Pituitary Cell Types

The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity.Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

MON-309 Prevalence of Silent Corticotroph Adenomas in a Large Cohort of Nonfunctioning Pituitary Adenomas: Plasma Proopiomelanocortin(POMC) Levels and Response to Pasireotide LAR

Silent corticotroph adenomas (SCAs) are tumors of the TPIT anterior pituitary cell lineage that do not lead to biochemical or clinical Cushings syndrome. Thus, they present as clinically nonfunctioning pituitary adenomas (CNFPAs) and are only diagnosed pathologically. Since they are often aggressive tumors, identification of a peripheral blood marker of SCA activity would be useful for diagnosis and monitoring. Some data suggest that aberrant processing of POMC, the precursor of ACTH, underlies the lack of elevated biologically active ACTH and thus cortisol excess in SCAs. We hypothesized that these tumors could secrete POMC, resulting in elevated plasma levels of POMC in patients with SCAs. Therefore, we investigated plasma POMC levels as a potential marker of this tumor type and correlated with tumor ACTH immunoreactivity (IR), which may be detecting unprocessed POMC. We studied 267 patients (134M, 133F, age 56.3±13.6yr) with CNFPAs (Cushings excluded) prior to surgery and at enrollment in a prospective, observational study. We also studied 9 patients with known SCAs with residual macroadenomas after surgery. Peripheral blood was sampled for POMC, ACTH and cortisol levels. POMC was measured by in-house two-site ELISA (detects POMC and 22kD pro-ACTH) and ACTH and cortisol by Immulite(Siemens). POMC levels were compared to the 95%CI of the mean of 70 healthy subjects and considered elevated if ≥ the 97.5 percentile of 39 fmol/mL. Of the CNFPA cohort, 12/267 had elevated POMC levels (range 39-166 fmol/mL) and 4 of the 12 had elevated ACTH levels (> 50 pg/ml)(range 53.6-76 pg/ml). Of the 12 with elevated POMC, 9 underwent surgery and 6 of them had positive ACTH IR in their tumors. An additional 13 patients in the CNFPA cohort had weak ACTH IR tumors, but POMC <39 fmol/mL and normal ACTH. POMC levels were elevated in all 9 known SCA patients (range 40-996 fmol/mL), being highest in those with the most aggressive tumors. ACTH levels were elevated in 6 of them (range 50.6-397 pg/ml). POMC levels were lowered with surgery in 4 of 4 SCA patients followed longitudinally. Two SCAs were treated with monthly pasireotide LAR (40 mg escalated to 60 mg) for 8 months. Plasma POMC levels fell from 104 to 21 fmol/mL in 1 patient, but did not change in the 2nd, 124 to 112 fmol/mL. Both patients had no change in the size of their macroadenomas during treatment. In summary, patients with elevated plasma POMC levels were correctly identified as SCAs in most surgically treated patients and POMC levels were lowered by tumor removal in all who were tested longitudinally. Although POMC levels were not elevated in some other patients with weakly positive ACTH IR tumors, further characterization of these tumors by lineage specific transcription factors is underway to confirm them to be of corticotroph cell origin. These data suggest that plasma POMC measurements may have clinical utility in the evaluation of SCAs and this warrants further study.

Transcription factor Creb3l1 regulates the synthesis of prohormone convertase enzyme PC1/3 in endocrine cells.

Transcription factor cAMP responsive element‐binding protein 3 like 1 (Creb3l1) is a non‐classical endoplasmic reticulum stress molecule that is emerging as an important component for cellular homeostasis, particularly within cell types with high peptide secretory capabilities. We have previously shown that Creb3l1 serves an important role in body fluid homeostasis through its transcriptional control of the gene coding for antidiuretic hormone arginine vasopressin in the neuropeptide‐rich magnocellular neurones of the supraoptic nucleus. In response to osmotic stimuli such as dehydration, vasopressin magnocellular neurones undergo remarkable transcriptome changes, including increased Creb3l1 expression, to ensure that the supply of vasopressin meets demand. To determine where else Creb3l1 fits into the secretory cell supply chain, we performed RNA‐sequencing of Creb3l1 knockdown anterior pituitary mouse corticotroph cell line AtT20. The target chosen for further investigation was Pcsk1, which encodes proprotein convertase enzyme 1 (PC1/3). PC1/3 is crucial for processing of neuropeptides and peptide hormones such as pro‐opiomelanocortin (POMC), proinsulin, proglucagon, vasopressin and oxytocin. Viral manipulations in supraoptic nuclei by over‐expression of Creb3l1 increased Pcsk1, whereas Creb3l1 knockdown decreased Pcsk1 expression. In vitro promoter activity and binding studies showed that Creb3l1 was a transcription factor of the Pcsk1 gene binding directly to a G‐box motif in the promoter. In the dehydrated rat anterior pituitary, Creb3l1 and Pcsk1 expression decreased in parallel compared to control, supporting our findings from manipulations in AtT20 cells and the supraoptic nucleus. No relationship was observed between Creb3l1 and Pcsk1 expression in the neurointermediate lobe of the pituitary, indicating a different mechanism of PC1/3 synthesis by these POMC‐synthesising cells. Therefore, Creb3l1, by regulating the expression of Pcsk1, does not control the processing of POMC peptides in the intermediate lobe.

Suprasellar extension independently predicts preoperative pituitary hormone deficiencies in patients with nonfunctioning pituitary macroadenomas: a single-center experience.

Nonfunctioning pituitary adenomas (NFPAs) are among the commonest benign tumors of the pituitary. Hypopituitarism is frequently present at the time of diagnosis, and this has been attributed to stalk portal vessel compression and/or destruction of normal anterior pituitary cells. The aim of our study was to examine possible factors at diagnosis associated with the presence of hypopituitarism. We retrospectively evaluated the records of patients with nonfunctioning pituitary macroadenomas from the database of our department. The inclusion criterion was the availability of imaging data regarding the extension of the lesion. A total of 148 patients (89 men, 60.1%) with nonfunctioning macroadenomas and available imaging data were identified. Mean age at diagnosis was 56.0 ± 14.5years, and hypopituitarism was found in 66.2%. The maximum diameter of the adenoma, the patient's age at diagnosis, and compression of the optic chiasm were significant factors predicting the presence of hypopituitarism (OR 1.077, p = 0.006; OR 1.025, p = 0.045; and OR 2.893, p = 0.042, respectively). Suprasellar adenomas with extension to adjacent sinuses, although larger than those with only suprasellar extension, did not differ as to the degree of hypopituitarism. Moreover, in suprasellar adenomas, prolactin levels, albeit not independently, were also related to hypopituitarism (OR 1.035, p = 0.045). In patients with NFPAs, prognostic factors related to increased risk of hypopituitarism are age at diagnosis, size of the adenoma, and most importantly the presence of suprasellar extension. These data accentuate the necessity for surgical decompression in case of suprasellar extension, in order, apart from saving visual acuity, to possibly avoid or reverse hypopituitarism.

MiR-7 mediates the signaling pathway of NE affecting FSH and LH synthesis in pig pituitary.

MicroRNA-7 (miR-7) is an important modulator of a plenty of gene expressions and the interrelated biological processes, highly expressed in porcine pituitary. Norepinephrine (NE), acting as an important neurotransmitter or/and a hormone secreted excessively under stress, affects the synthesis and secretion of various hormones, including pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are the key hormones which regulate sexual maturation and reproductive functions. However, the relationship among NE, miR-7 and gonadotropin needs to be elucidated. The aim of this study was to identify whether miR-7 involved in the NE-adrenoceptor signaling pathway affects the synthesis and secretion of FSH and LH in porcine pituitary. Our results showed that the NE intracerebroventricular injection increased pituitary miR-7 level and the synthesis and secretion of FSH and LH in porcine, whereas the inhibition of either endogenous miR-7 or β-adrenergic receptors hindered the rise of FSH and LH synthesis induced by NE in cultured primary porcine anterior pituitary cells. Further, we identified the molecular type of β-adrenergic receptors and the signaling pathway in porcine pituitary, and we found that NE played its roles relying on adrenoceptor beta 2 (β2AR) and the RAF/MEK/ERK1/2 signaling pathway. The phosphorylation of ERK1/2 upregulated miR-7 level which subsequently enhanced FSH and LH synthesis by targeting to Golgi glycoprotein 1 (GLG1). These suggest that miR-7 mediates NE's effect on promoting FSH and LH synthesis in porcine pituitary.

Gonadotropin-releasing hormone-Cu complex (Cu-GnRH) transcriptional activity in vivo in the female rat anterior pituitary gland

Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo.We analysed pituitary mRNA expression of Egr-1, Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP6–38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA.All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1, Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation.In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

Functional effect of laminin α chains on endocrine cells in rat anterior pituitary gland

Functional effect of laminin α chains on endocrine cells in rat anterior pituitary gland