Leptin: A Metabolic Signal for the Differentiation of Pituitary Cells

Pituitary cell function is impacted by metabolic states and therefore must receive signals that inform them about nutritional status or adiposity. A primary signal from adipocytes is leptin, which recent studies have shown regulates most pituitary cell types. Subsets of all pituitary cell types express leptin receptors and leptin has been shown to exert transcriptional control through classical JAK/STAT pathways. Recent studies show that leptin also signals through post-transcriptional pathways that involve the translational regulatory protein Musashi. Mechanistically, post-transcriptional control would permit rapid cellular regulation of critical pre-existing pituitary transcripts as energy states change. The chapter will review evidence for transcriptional and/or post-transcriptional regulation of leptin targets (including Gnrhr, activin, Fshb, Gh, Ghrhr, and Pou11f1) and the consequences of the loss of leptin signaling to gonadotrope and somatotrope functions.

The sodium leak channel NALCN encodes the major background sodium ion conductance in murine anterior pituitary cells

The pituitary gland, the so-called master gland produces and secretes a variety of hormones essential for regulating growth and development, metabolic homeostasis, reproduction, and the stress response. The interplay between the brain and peripheral feedback signals controls hormone secretion from pituitary cells by regulating the properties of ion channels, and in turn, cell excitability. Endocrine anterior pituitary cells fire spontaneous action potentials to regulate their intracellular calcium level and eventually hormone secretion. However, the molecular identity of the non-selective cationic leak channel involved in maintaining the resting membrane potential at the firing threshold remained unknown. Here, we show that the sodium leak channel NALCN, known to modulate neuronal excitability, also regulates excitability in murine anterior pituitary cells. Using viral transduction combined with electrophysiology and calcium imaging we show that NALCN encodes the major Na+ leak conductance which tunes the resting membrane potential close to firing threshold to sustain the intrinsically-regulated firing in endocrine pituitary cells. Genetic interruption of NALCN channel activity, hyperpolarised the membrane potential drastically and stopped the firing activity, and consequently abolished the cytosolic calcium oscillations. Moreover, we found that NALCN conductance forms a very small fraction of the total cell conductance yet has a profound impact on modulating pituitary cell excitability. Taken together, our results demonstrate that, NALCN is a crucial regulator of pituitary cell excitability and supports spontaneous firing activity to consequently regulate hormonal secretion. Our results suggest that receptor-mediated and potentially circadian changes in NALCN conductance can powerfully affect the pituitary activity and hormone secretion.

The bromodomain inhibitor JQ1+ reduces calcium-sensing receptor activity in pituitary cell-lines

Corticotrophinomas represent 10% of all surgically removed pituitary adenomas, however, current treatment options are often not effective and there is a need for improved pharmacological treatments. Recently, JQ1+, a bromodomain inhibitor that promotes gene transcription by binding acetylated histone residues and recruiting transcriptional machinery, has been shown to reduce proliferation in a murine corticotroph cell-line, AtT20. RNA-Seq analysis of AtT20 cells following treatment with JQ1+ identified the calcium-sensing receptor (CaSR) gene as significantly downregulated, which was subsequently confirmed using real-time PCR and western blot analysis. CaSR is a G protein-coupled receptor that plays a central role in calcium homeostasis but can elicit non-calcitropic effects in multiple tissues, including the anterior pituitary where it helps regulate hormone secretion. However, in AtT20 cells, CaSR activates a tumour-specific cAMP pathway that promotes ACTH and PTHrP hypersecretion. We hypothesised that the Casr promoter may harbour binding sites for BET proteins, and using chromatin immunoprecipitation (ChIP)-sequencing demonstrated that the BET protein Brd3 binds to the promoter of the Casr gene. Assessment of CaSR signalling showed that JQ1+ significantly reduced Ca2+e-mediated increases in intracellular calcium (Ca2+i) mobilisation and cAMP signalling. However, the CaSR negative allosteric modulator, NPS-2143, was unable to reduce AtT20 cell proliferation, indicating that reducing CaSR expression rather than activity is likely required to reduce pituitary cell proliferation. Thus, these studies demonstrate that reducing CaSR expression may be a viable option in the treatment of pituitary tumours. Moreover, current strategies to reduce CaSR activity, rather than protein expression for cancer treatments, may be ineffective.

Two Cases of Pembrolizumab-Induced Secondary Adrenal Insufficiency

Title: Two cases of pembrolizumab-induced secondary adrenal insufficiency Background: Although few cases regarding pembrolizumab-induced secondary adrenal insufficiency were reported, it has recently been published that significantly higher prevalence of human leucocyte antigen (HLA)-DR15, B52, and Cw12 was revealed in 11 Japanese patients with immune checkpoint inhibitor-induced secondary adrenal insufficiency as compared with healthy controls and DR15 might be a predictive marker. This is based on the evidence that DR15 has associations with IL-17-mediated autoimmune disease such as Hashimoto’s thyroiditis, and that anti-programmed death 1 (PD-1) inhibitors activate the Th1 and Th17 pathways. We here introduce two Japanese patients with pembrolizumab-induced secondary adrenal insufficiency from a viewpoint of their HLA typing test results. Clinical Case: Case 1. A 52-year old male diagnosed with stage IV lung cancer (squamous non-small cell lung cancer) was treated with chemotherapy consisting of carboplatin, nab-paclitaxel, and pembrolizumab. Six days after the fourth cycle of pembrolizumab, he suffered from hypoglycemia and hyponatremia. Endocrinological examination findings showed ACTH and cortisol deficiency throughout the day, after which a diagnosis of secondary adrenal insufficiency was made based on no response shown in insulin tolerance, anterior pituitary function, and rapid ACTH stimulation tests. Pituitary cell antibody-1 was negative, and no evidence of a swollen/atrophied pituitary gland or space occupying lesion was observed in magnetic resonance imaging (MRI) results. Furthermore, Hashimoto’s thyroiditis was suspected based on anti-thyroid peroxidase antibody positivity. HLA typing test results highly corresponded with the reported results (A24, B52, Cw12, DR4, DR15). Case 2. A 70-year old male was treated with pembrolizumab for recurrence of lung squamous cell carcinoma (stage IV). Two weeks after completion of five cycles, severe diarrhea occurred, suspected to be a side effect of pembrolizumab, which was relieved with temporary use of prednisolone. However, diarrhea recured accompanied with a high fever, eosinophilia, and acute hyponatremia. Based on results showing deficiency of ACTH and cortisol, adrenal insufficiency was suspected. Anterior pituitary function and rapid ACTH stimulation test results supported that diagnosis, while pituitary cell antibody-1 was negative and MRI findings were not remarkable. Notably, completely different HLA typing test results were observed. Conclusion: Similar to the previous report, HLA-A24, B52, Cw12, DR4, and DR15 were found in case 1, whereas all subtypes including HLA-DR15 were lacking in case 2. We think that it should not be given low priority for cases lacking DR15 and the possibility of an association with IL-17 in such cases is required in feature study.

The Musashi1 RNA-Binding Protein Functions as a Leptin-Regulated Enforcer of Pituitary Cell Fate and Hormone Production

The pituitary gland is the major endocrine organ that produces and secretes hormones in response to hypothalamic signals to regulate important processes like growth, reproduction, and stress. The anterior pituitary adapts to metabolic and reproductive needs by exhibiting cellular plasticity, resulting in altered hormone production and secretion. The adipokine, leptin, serves a critical role to couple energy status to pituitary function. We have recently reported that the cell fate determinant, Musashi, functions as a post-transcriptional regulator of target mRNA translation in the mouse pituitary and have speculated that Musashi may modulate pituitary cell plasticity. However, the underlying mechanisms governing such pituitary plasticity are not fully understood. Musashi is an mRNA binding protein that is required for self-renewal, proliferation, and to control the differentiation of stem and progenitor cells. We have recently shown that Musashi is expressed in Sox2+ pituitary stem cells and surprisingly, we also found Musashi expression in all differentiated hormone expressing cell lineages in the adult anterior pituitary. The role of Musashi in these mature differentiated cells is unknown. We have observed that a range of critical pituitary mRNAs, including the lineage specification transcription factors Prop1 and Pou1f1, as well as hormone mRNAs including Tshb, Prl, and Gnrhr, all contain consensus Musashi binding elements (MBEs) in their 3’ untranslated regions (3’ UTRs). Using RNA electrophoretic mobility shift assays (EMSAs) and luciferase mRNA translation reporter assays we show that Musashi binds to these mRNAs and exerts inhibitory control of mRNA translation. Moreover, we determined that leptin stimulation opposes the ability of Musashi to exert translational repression of the Pou1f1 and Gnrhr 3’ UTRs. This de-repression does not require regulatory phosphorylation of Musashi on two conserved C-terminal serine residues. Interestingly in the same cell assay system, Musashi exerts translational activation of the Prop1 3’ UTR. We observed that this translational activation requires Musashi phosphorylation on the two regulatory C-terminal serine residues, consistent with the requirement for regulatory phosphorylation to drive translational activation of Musashi target mRNAs during Xenopus oocyte cell maturation. The distinction between MBEs in 3’ UTRs that exert repression (Pou1f1, Prl, Tshb, and Gnrhr) and the Prop1 3’ UTR that directs translational activation is under investigation. We propose that Musashi acts as a bifunctional regulator of pituitary hormone production and lineage specification and may function to maintain pituitary hormone plasticity in response to changing organismal needs.

Single Nucleus Transcriptome and Chromatin Accessibility Landscapes of Human Pituitaries

The pituitary gland regulates key physiological functions, including growth, sexual maturation, reproduction, and lactation. Here, we present a paired single-nuclei (sn) transcriptome and chromatin accessibility characterization of six post-mortem human pituitaries. These samples were from juvenile, adult, and elderly male and female subjects. Well-correlated snRNAseq and snATACseq datasets facilitated robust identification of the major pituitary cell types in each sample. Using latent variable pathway analysis, we uncovered previously unreported coordinated gene expression modules and chromatin accessibility programs for each major cell type as well as an age-specific program across all the endocrine cell types. These largely appear to be congruent between human and mouse datasets. Given the importance of murine models in the study of human pituitary disorders and pituitary physiology, we next sought to compare expression profiles of pituitary cell types in mouse vs. human. Murine and human cell types were well correlated, exemplified by coordinated gene expression programs, especially for undifferentiated stem cells (SCs). In both species, we identified clusters corresponding to naive and committing SCs. All human SC clusters expressed the established SC markers SOX2 and SOX9, as well as genes involved in SC regulatory pathways (WWTR1, YAP1 andPITX2). Additional markers previously reported in murine pituitary SCs were also found in human SC, including WIF1, LGR5, FOS, CDH1, EGFR, LGR4, and WLS. Remarkably, in human, the main naive SC cluster was roughly divided into a high-JUN and a low-JUN expressing subgroup, whereas Jun expression was less pronounced in the murine SC cluster. In both species, committing SC clusters expressed the endocrine markers for POU1F1, TSHB, or POMC, while SCs committing to an intermediate lobe/melanotrope cell identity were distinguishable based on PAX7 expression. In addition, in the human datasets we identify a population of cells as originating from the pars tuberalis. We offer a range of markers that can be utilized for in vivo validation of these cells. Overall, the characterization of the murine and human pituitary SCs strongly suggests the co-existence of subpopulations with different lineage commitments in addition to a single uncommitted SC population. This sn atlas of the human pituitary is a valuable resource that will be made web-accessible.

Long extensions with varicosity-like structures in gonadotrope Lh cells facilitate clustering in medaka pituitary culture

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhβ protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.