Pituitary Cells Research Articles

RF13 | PMON143 BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors

Abstract Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

LBODP078 Role Of Ire1a-xbp1 Pathway In Rodent Functional Pituitary Tumor Cells.

Abstract Introduction Pituitary tumors (PTs) occur in almost 17% of the population and they represent about 10% of all intracranial tumors. At present, many patients with pituitary tumors suffer from symptoms and complications caused by abnormal hormone production and/or mass effects due to limited treatment options. Therefore, the development of novel therapies is an urgent issue. The inositol requiring enzyme 1a (IRE1a)-X-box binding protein 1 (XBP1) pathway is an important component for the endoplasmic reticulum (ER) stress response. This pathway is activated by immature protein overload in the ER and elimination or repair of such protein can reduce ER stress. This pathway has been reported to regulate cancer cell growth and secretory protein production. The detection of potential XBP1 binding sites in the promoter region of rat GH and mouse POMC similar to those of human genes suggests the involvement of the IRE1a-XBP1 pathway in the production of these hormones and pituitary tumor cell growth. In this study, we aimed to clarify the role of the IRE1a-XBP1 pathway in functional pituitary tumor cells. Methods and Results We conducted in vitro experiments by using rat GH-producing pituitary GH4 cells and mouse ACTH-producing AtT-20 cells as the models of GH- and ACTH-producing pituitary adenomas, respectively. Cell proliferation was measured by WST-8 assay, gene expression levels by qPCR, and protein expression by western blotting, respectively. In GH4 cells, STF08310 (40µM), an IRE1a inhibitor, reduced levels of GH and XBP1 spliced form (XBP1s) mRNA and GH protein with suppressed cell proliferation. In AtT-20 cells, STF08310 (40µM) also suppressed protein expression of proopiomelanocortin (POMC) and cell proliferation. Gene suppression of IRE1a by siRNA and over-expression of XBP1s showed marked reduction and increase of POMC protein, respectively. Conclusion Our findings suggest that the IRE1a-XBP1s pathway would be a possible therapeutic target that has both anti-tumoral and hormone control effects against functional pituitary tumor. Presentation: No date and time listed

PMON148 ACQUIRED TEMOZOLOMIDE RESISTANCE IN CORTICOTROPE PITUITARY TUMOR CELLS

Abstract Introduction Temozolomide (TMZ) treatment is an effective treatment for aggressive pituitary tumors, leading to tumor regression or stabilization of tumor progression in the majority of cases. In cases of disease recurrence, however, TMZ treatment does not control tumour regrowth. The expression of O6-methyl-guanine-DNA-methyltransferase (MGMT) protein and mismatch repair components (MMR) are implicated in the mechanism of TMZ resistance. In the present study, we aimed to develop an in vitro model of acquired TMZ resistance and to describe its mechanism. Methods The mouse AtT20 corticotrope pituitary tumor cell line was used. Two protocols were developed, one based on high dose of TMZ (high TMZ-challenge) and another one using lower TMZ concentrations (low TMZ-challenge) that are clinically achievable after a standard TMZ schedule. Both protocols consisted in pulse exposures to increasing concentrations of TMZ for 24rhs. TMZ concentrations in the high TMZ-challenge were 100, 200 and 300 µM while in the low TMZ-challenge were 20, 40, 60 and 80 µM. AtT20 cells treated with challenges of the vehicle dimethyl sulfoxide (vehicle-challenged cells) were used as control. Results The TMZ challenges induced a significant increase in EC50 (decreased sensitivity) of the growth inhibitory effect of TMZ in TMZ-challenged cells compared to vehicle-challenged cells in both protocols (high TMZ-challenge, EC50 = 123 µM vs 19 µM, p<0.001; low TMZ-challenge, EC50 = 69 µM vs 21 µM, p<0.001). The TMZ-challenged cells remained resistant compared to vehicle-challenged cells even after 9 weeks following the last TMZ challenge. The apoptotic cell death was higher in vehicle-challenged cells compared to TMZ-challenged cells in both protocols, albeit not statistically significant in the low TMZ-challenge protocol (high TMZ-challenge, p=0.003; low TMZ-challenge, p=0.169). TMZ-induced changes in cell cycle phases were more significant in vehicle-challenged cells compared to TMZ-challenged cells. Differences in cell morphology were observed between vehicle- and TMZ-challenged cells in the high dose protocol. In vehicle-challenged cells, small rounded and fusiform-shaped cells were observed, whereas in the TMZ-challenged cells only rounded cells were detected in culture. mRNA expression of MGMT was significantly higher in TMZ-challenged cells compared to vehicle-challenged cells. MMR components expression was not affected by TMZ challenges. Conclusion The present study describes an acquired TMZ-resistant corticotrope pituitary tumor cell model. TMZ resistance was demonstrated in both protocols by a sustained increase in TMZ EC50, small TMZ-induced cell cycle changes, and lower increase in TMZ-induced apoptosis. Additionally, TMZ challenges affected AtT20 cell morphology. This was evident only in the high TMZ-challenge protocol. Acquired TMZ resistance was associated with a strong increase in MGMT expression. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF13 | PMON146 Molecular determinants of cavernous sinus invasion by pituitary adenomas: functional in vitro studies and in vivo investigations in a rat model of invasive pituitary tumorigenesis obtained by stereotactic pituitary GC cells injection

Abstract Context Surgical removal is the primary treatment option for pituitary adenomas. However, pituitary surgery is frequently incomplete because of invasion of extrasellar cerebral structures. In particular, the vicinity with the carotid artery hinders the radical surgical excision of the adenoma portion invading the cavernous sinus. Our objective was to study the molecular bases of the cavernous sinus invasion by pituitary adenomas. Methods We obtained a unique tissue collection of 19 invasive pituitary adenomas with a sample from the intrasellar portion and a sample from the portion invading the cavernous sinus for each adenoma. We used RNA-sequencing to compare the gene expression patterns of the invading and intrasellar portions. The implication of one differentially expressed candidate gene in the invasive behavior was first analyzed in vitro using Transwell Assay to study the impact of its pharmacologic inhibition on cell migration and invasion. These experiments were conducted in murine lactosomatotroph GH3 cells and gonadotroph LbT2 cell. To further study the role of this gene on tumor growth and behavior in vivo, we elaborated a model of invasive pituitary adenomas by stereotactic injection of murine somatotroph GC cell into the lobes of the pituitary gland of female Wistar Furth rats. Twelve rats (10-12 week-old) received 20.000 GC cells in each pituitary lobe. After surgery, rats were monitored weekly, tumor development was assessed fortnightly by 3 successive 7Tesla MRI and analyzed by Horos software. Rats were sacrificed 7 weeks after GC cells injection. Six of the 12 rats were treated during this period with the pharmacological inhibitor of the candidate gene. Results RNA-sequencing identified 159 up-regulated genes and 11 down-regulated genes in the invasive adenoma portion. In vitro pharmacological inhibition of the candidate gene decreased cell migration and invasion in GH3 cells (p=0.0205 and p=0.0038) and LbT2 cell (p=0.0345 and p=0.0131). Amongst the 12 injected animals, 11 (92%) rats developed invasive pituitary tumors. Tumor growth was rapid, causing death from intracranial hypertensions before the theoretical end of the study protocol in 7 animals. Pharmacological inhibition tended to slow tumor growth from 30.3 mm3/week to 7.8 mm3/week (p=0.12) and decreased cumulative mortality (83% in untreated animals versus 33% in treated animals, p=0.08). Conclusion We described the molecular signature associated with the invasive behavior of pituitary adenomas and identified a therapeutic target, which is related to pituitary cells migration and invasion in functional in vitro studies. Pharmacologic inhibition of this target tended to decrease tumor growth and mortality from intracranial hypertension in vivo, however larger numbers of animals are necessary to confirm this observation. Our original approach of orthotopic cell injection into rat pituitaries resulting in tumor development provides a new tool for molecular studies of pituitary tumorigenesis and for the screening of new therapeutic agents. Presentation: Sunday, June 12, 2022 1:06 p.m. - 1:11 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF01 | PMON46 Regulation of the Pituitary Cell Lineage Regulator Prop1 by the Stem Cell Determinant Musashi

Abstract The pituitary gland controls responses to changing physiological requirements during growth, pregnancy, puberty, and stress. Synthesis and secretion of appropriate pituitary hormones in response to changing organismal demands is mediated through a remarkable capacity for cellular plasticity of the distinct hormone-producing cells. The mechanisms governing pituitary cellular plasticity are not fully understood. We hypothesize that plasticity in the adult tissue is mediated through similar mechanisms to those that occur during embryonic and postnatal pituitary development. The Prophet of Pit1(Prop1), a paired-like homeodomain transcription factor, is the earliest pituitary-specific transcription factor and is highly expressed in pituitary progenitor cells where it controls lineage commitment to the distinct hormone-producing cell types. Our recent work has identified post-transcriptional regulation of pituitary gene expression in the adult tissue via the stem cell determinant, mRNA binding protein, Musashi. The full-length mouse Prop1 (mProp1) mRNA 3' untranslated region (UTR) contains 24 consensus binding elements for Musashi (MBEs) and demonstrates Musashi1-dependent translational activation in vitro. Specifically, the terminal mProp1 3' UTR MBE directs 40% of Musashi1-mediated translation activation, and mutational disruption of this terminal MBE abolishes all Musashi1-dependent translational activation. Immunoprecipitation of Musashi-mRNA complexes from adult mouse pituitaries confirmed Musashi1-mProp1 mRNA association in adult tissue. The gene sequence data for the human Prop1 (hPROP1) mRNA 3' UTR is incomplete in the Ensembl and RefSeq databases. Using published RNA sequencing datasets, we identified an expressed full-length hPROP1 mRNA 3' UTR of 6996 nucleotides with 54 predicted consensus MBEs, suggesting that the human PROP1 mRNA, like the murine Prop1 mRNA, may be subject to Musashi-dependent translational control. Our findings are consistent with the hypothesis that the Musashi protein exerts evolutionarily conserved control of Prop1 mRNA translation to facilitate plasticity of anterior pituitary function. Presentation: Saturday, June 11, 2022 1:31 p.m. - 1:36 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

PMON170 Treating human primary non-functioning pituitary adenoma cells with cabergoline

Abstract Clinically non-functioning pituitary adenomas (NFPAs) represent the second most common subtype of pituitary tumours, representing 15-43% of all pituitary adenomas. NFPAs are usually benign tumours with no clinical evidence of hormonal hypersecretion. In most of the cases the tumours are arising from the gonadotroph linage, but silent corticotroph, somatotroph, thyrotroph and rarely lactotroph cell characteristics can be discovered with immunostaining. As a result of lack of hormonal hyper-secretion, the diagnosis of NFPAs is made most often when the patient presents with mass effects due to a macroadenoma. NFPAs are most common tumour types in surgical series, and their primary treatment is indeed surgery. However, complete resection is achieved only in about 66% of the cases, and 20% of gross total resected tumours recur after 10 years. Over the past few decades, remarkable progress has been achieved regarding medical treatment options for pituitary tumours. However, NFPAs remain the only subtype with no widely accepted pharmacological treatment. Dopamine receptor type 2 (DRD2) and somatostatin receptor (SST) expression have been demonstrated in NFPAs, prompting the investigation of dopamine agonists and somatostatin analogues as potential treatment strategies. The DRD2 agonist cabergoline is already used as first-line treatment of prolactinomas to induce tumour shrinkage and reduce prolactin secretion. Here we aim to investigate the efficacy of cabergoline on human NFPA tissue. We assessed DRD2 expression levels via immunohistochemistry and qPCR in a large cohort of NFPAs (n=40) and in few prolactinomas (n=10). D2DR has two different isoforms, long (D2RL) and short (D2RS), which are differently expressed and can induce different effects on cell viability. In cabergoline-sensitive prolactinomas D2RS is the predominant isoform. NFPAs shown 10-fold and 5-fold decrease expression of D2RS and D2RL compared to prolactinomas, respectively. Additionally, in NFPAs we observed a significant decrease of cAMP production after cabergoline treatment (45% ±7; p<0.0001); however, viability after one-week cabergoline treatment showed only 4% (±0.7; p<0.0002) reduction, compared to a 10% decrease in prolactinomas (p<0.05). We also observed no difference in secretion of chromogranin A release in NFPAs upon treatment with cabergoline. We have also seen in NFPAs downregulation of Ga i2 protein expression levels, highlighting the general decrease of expression levels in the dopamine pathway (50% less, p<0.004). Our data suggest that the difference in cabergoline responses between NFPAs and prolactinomas may be due to their distinct expression of D2DR isoforms, which differ by 29 amino acids in the third cytoplasmic loop, essential for G-protein binding. It has been shown that D2RL, but not D2RS, requires Gai2. The difference in D2R isoform expression could translate to distinct G-protein activation, ultimately leading to the contrasting viability results after cabergoline treatment. Taken together, these data will help inform future treatment strategies for patients with NFPAs. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

PMON171 DRD2 Agonist Cabergoline Abolished the Escape Mechanism Induced by mTOR Inhibitor Everolimus in Pituitary Neuroendocrine Tumoral Cells

Abstract PI3K-AKT-mTOR inhibitors have been proposed as alternative therapeutic strategy for tumors resistant to conventional drugs. The mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to display antiproliferative effects on a wide spectrum of tumors. In vitro studies demonstrated that everolimus inhibited pituitary neuroendocrine tumors (PitNETs) cell growth and vitality in a subset of patients. Sensitivity to everolimus has been demonstrated to be reduced by an escape mechanism that increases AKT phosphorylation (p-AKT), leading to pro-survival pathway activation. This limited efficacy of everolimus due to AKT re-activation could be overtaken by upstream AKT phosphorylation inhibition. Given it was recently demonstrated that DRD2 mediates a reduction of p-AKT in a subset of NF-PitNETs and in rat prolactin-secreting tumor cells MMQ through a β-arrestin 2-dependent mechanism, the aim of the present work was to test the efficacy of adjuvant treatment with cabergoline in increasing sensitivity of human primary cultured NF-PitNET cells to antiproliferative effects of everolimus. In addition, to test a possible role of β-arrestin2. We found that 9 out of 14 NF-PitNETs were resistant to everolimus 1 nM, but the combined treatment with cabergoline inhibited cell proliferation in 7 out of 9 tumors (-31.4 ± 9.9%, p < 0.001 vs basal), increased p27 and reduced cyclin D3 expression. In everolimus unresponsive NF-PitNETs group, 3 h everolimus treatment determined a significant increase of p-AKT/total-AKT ratio (+2.1-fold, p < 0.01, vs basal), and this effect was reverted by cabergoline cotreatment in all tested samples. To investigate the molecular mechanism involved, we used MMQ cells as a model of everolimus escape mechanism. Indeed, 1nM everolimus did not affect MMQ cells proliferation due to an increase of p- AKT/total-AKT ratio (+1.53 ± 24%, p<0.001 vs basal), whereas cabergoline reduced cell proliferation (-22.8 ± 6.8%, p<0.001 vs basal) and AKT phosphorylation. The combined treatment of everolimus and cabergoline induced a significant reduction of both cell proliferation (-34.8 ± 18%, p<0.001 vs basal) and p-AKT/total-AKT ratio (-34.5 ± 14%, p<0.001 vs basal). To test a possible involvement of β-arrestin 2, silencing experiments were performed in MMQ cells. Our data showed that the lack of β-arrestin 2 prevented everolimus and cabergoline co-treatment inhibitory effects on both p-AKT and cells proliferation. In conclusion, these data revealed that cabergoline might overcome the everolimus escape mechanism in NF-PitNETs and tumoral lactotrophs, by inhibiting upstream AKT activation. The co-administration of cabergoline might improve mTOR inhibitors antitumoural activity, paving the way for a potential combined therapy in β-arrestin 2 expressing NF-PitNETs or other PitNETs resistant to conventional treatments. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP335 Mutual Effects of Orexin and BMPs on Gonadotropin Expression by Mouse Gonadotrope LβT2 Cells

Abstract Orexins are neuropeptides that express primarily in the hypothalamus and are produced in two isoforms, orexin A and orexin B. There are two kinds of G protein coupled receptors, orexin type 1 (OX1R) and type 2 (OX2R) receptors. Orexin has been reported to have key roles on sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine organs, and orexin affects the regulation of the endocrine system. In our previous experiments, we have revealed the impact of orexins on anterior pituitary functions. For instance, we reported that orexin A plays an inhibitory role in prolactin production through the suppression of endogenous bone morphogenetic protein (BMP) activity in rat pituitary lactotrope GH3 cells. It was also reported that orexin A enhances pro-opiomelanocortin (POMC) transcription by upregulating corticotropin-releasing hormone (CRH) receptor signaling and by downregulating BMP-Smad signaling in mouse corticotope AtT20 cells. However, the effects of orexin on the endocrine function regarding gonadotrope cells remain unclear. We have recently uncovered that core clock genes and BMPs have mutual effects on luteinizing hormone (LH) expression in a phase-dependent manner by mouse gonadotrope LβT2 cells. In the present study, we investigated the effect of orexin on pituitary gonadotropin expression using LβT2 cells, which express OX1R and OX2R, by focusing on the functional involvement of BMP system and clock genes. It was revealed that orexin A stimulation increased LHβ and FSHβ mRNA expression in a concentration-responsive manner in the absence of GnRH, and interestingly, GnRH co-treatment further upregulated LHβ mRNA expression in LβT2 cells. Regarding the interrelationships between the signalings of orexin and BMPs, it was also revealed that orexin A pretreatment enhanced the BMP receptor signaling detected as the Smad1/5/9 phosphorylation, indicating that orexin enables to upregulate the BMP actions in LβT2 cells. In our previous studies, we reported that several BMP ligands such as BMP-6, -7 and 15 expressed in LβT2 cells can promote gonadotropin transcription, in which BMP-6 regulates GnRH-stimulated LH expression by modulating the sensitivity to somatostatin analogs. Based on the present results, it was implied that endogenous orexin can be functionally involved in the underlying mechanisms of gonadotropin expression. Collectively, orexin enhances gonadotropin expression by regulating BMP signaling in gonadotrope cells. Here, we will also discuss functional involvement of clock genes in the regulatory system of gonadotropin secretion induced by orexin and BMPs. Presentation: No date and time listed

OR08-2 Functional Association of the Stem Cell Protein Musashi With LSM14B in Control of mRNA Translation

Abstract The Musashi RNA-binding protein functions as a gatekeeper of cell maturation and maintains stem cell plasticity by regulating the translation of target mRNAs. The adult anterior pituitary tissue expresses a high level of Musashi and also demonstatres a high level of cell plasticity, indicating a Musashi-dependent function in maintaining endocrine homeostasis in the anterior pituitary. Towards understanding the mechanism(s) by which Musashi functions to control cell plasticity, we have identified co-associated proteins necessary for Musashi function. The two Musashi isoforms (Musashi1 and Musashi2) are conserved across species and are structurally characterized by two N-terminal RNA-recognition motifs (RRMs, that associate with specific sequences in the 3' untranslated region of mRNAs targets) and a disordered C-terminus. Musashi uses these domains to selectively repress, or to activate, mRNA translation in a manner specific to target mRNA and cell context. Both Musashi isoforms contain two C-terminal regulatory serine residues that require phosphorylation to enable mRNA translational activation. However, the Musashi proteins do not have inherent mRNA translation function and utilize co-associated protein complexes to mediate mRNA translation. We have previously identified Musashi1 binding protein partners through mass spectrometry. Here, we have identified the protein interactions that are required for Musashi1-dependent translational activation. Knockdown of target protein levels in Xenopus laevis oocytes indicated that the PABP4, CELF2, LSM14A/B, ELAVL1, ELAVL2, ELAVL4, and PUM1 proteins are required for Musashi target mRNA translational activation. We further determined that the effect of LSM14A/B dual knockdown was in fact specific to LSM14B alone. LSM14B is an RNA-binding protein that is required for RNA granule formation, RNA transcript metabolism, and mRNA translation. Studies in mouse oocytes have shown that LSM14B is required to maintain transcript levels of the Musashi1 target Cyclin B1 (Ccnb1) mRNA prior to mRNA translation. In the adult murine pituitary, LSM14B is ubiquitously expressed in both hormone-producing cells and in stem cells as indicated through single cell RNA sequencing. These findings indicate that LSM14B and Musashi1 may function cooperatively to regulate pituitary hormone production and cell differentiation in response to changes in physiological demand. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

PMON29 A Comparative Analysis of Musashi-Dependent Control of Mouse and Human Pituitary mRNA Translation

Abstract Specialized cell lineages within the anterior pituitary produce specific hormones which, in turn, regulate diverse biological processes ranging from growth, metabolism, and reproduction to stress. Interestingly, these specialized cell lineages exhibit a high degree of cellular plasticity, readily altering their differentiation state to produce different hormones in response to organismal demand. Underlying molecular mechanisms controlling cellular plasticity in the pituitary are not fully defined but appear to involve post-transcriptional regulation exerted by the Musashi family of sequence-specific RNA binding proteins. Musashi is a bifunctional regulator of target mRNAs and can exert either repression or activation of translation in a context-dependent manner. Our recent work has implicated a role for Musashi-dependent repression of key pituitary mRNAs in the mouse, including a lineage-specific transcription factor (Pou1f1), the gonadotropin releasing hormone receptor (Gnrhr), and two anterior pituitary hormones (Prl and Tshb). In this study, we look to determine the potential relevance of Musashi-dependent mRNA translational control to the human pituitary. Reflecting our findings in the adult mouse, a re-analysis of published human fetal pituitary single cell RNA sequencing data revealed Musashi1 (MSI1) and Musashi2 (MSI2) expression in both stem/progenitor cell populations as well as in all hormone-producing cell lineages of the anterior pituitary. We further report that the human homologs of identified Musashi-target mRNAs in the mouse pituitary also possess Musashi binding elements (MBEs) in their 3' untranslated regions (UTRs). In the case of the human Pou1f1 3' UTR, we were able to verify Musashi-dependent mRNA translational repression in reporter assays as seen with the murine Pou1f1 3' UTR. Interestingly, a germline Pou1f1 mutation identified in human patients, disrupts a consensus MBE in the human Pou1f1 3' UTR and abolishes Musashi-dependent repression. We developed a bioinformatic pipeline to characterize pathogenic human mutations catalogued in the Geno2MP database for disruption or creation of consensus MBEs in pituitary-specific mRNA 3' UTRs. Our findings suggest an evolutionarily conserved role for Musashi in the post-transcriptional regulation of pituitary mRNAs in both mice and humans and identify several human mutations which may perturb Musashi regulation of human pituitary function. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP289 A rare case of COVID-19 vaccine-associated pituitary apoplexy

Abstract Background The occurrences of pituitary apoplexy following COVID-19 vaccination had only been reported twice. We present the case of a 24-year-old female who developed pituitary apoplexy following the second dose of an AstraZeneca COVID-19 vaccine. Clinical Case A 24-year-old female presented with sudden onset of severe frontal headache, a day after receiving her second AstraZeneca vaccine dose. She denied other symptoms including blurring of vision, eye pain and diplopia. On admission, her blood pressure was 120/80 mmHg, with no evidence of postural hypotension. She was overweight with a BMI of 29kg/m2. Confrontational visual field assessment was normal with no evidence of bitemporal hemianopia. An MRI of the pituitary showed a heterogenous enhancement of the pituitary gland, extending to the suprasellar region, measuring approximately 1.1cm×1.6×1.9cm (AP×W x CC) with features suggestive of pituitary apoplexy with mass effect to the optic chiasm and cavernous portion of the left ICA. Pituitary hormonal tests results showed normal anterior pituitary hormone levels, with the exception of insulin-like growth factor-1 (IGF-1) at a level of 417 ng/ml. Overnight dexamethasone test was performed and showed suppressed cortisol level of 48.4 nmol/L. Growth hormone suppression test resulted in suppressed GH level of <0.1ng/ml in the presence of hyperglycemia. Pituitary hypophysitis was considered because of the more likely age group and the typical presentation of severe headache. In the context that the headache had started shortly after the administration of the second dose of COVID-19 vaccine, this potentially could have been the trigger for the occurrence of pituitary apoplexy She was treated with high dose steroids and the headache improved. A follow up pituitary MRI showed a significant 50% reduction in the size of the pituitary mass. To date, this report would be the third linking COVID-19 vaccination and pituitary apoplexy. The first one occurred in a 44-year-old man, who presented with sudden onset of blurring of vision 3 days following the second dose of COVID-19 vaccine. His MRI showed a suprasellar mass with optic chiasm. His endoscopic transsphenoidal resection of the pituitary tumor showed focal hemorrhage and necrosis of pituitary adenoma cells. The second case occurred in 37 years old lady 5 days following the dose of Astra Zeneca vaccine. MRI finding showed consistent with pituitary apoplexy . Conclusion This report highlights a rare complication of the COVID-19 vaccine that could cause pituitary apoplexy. Possible explanation could be due to vascular dysfunction secondary to the overwhelming immunological response. Reference Jaggi, S. and S. Jabbour, Abstract #1001394: A Rare Endocrine Complication of the COVID-19 Vaccine. Endocrine Practice, 2021. 27(6): p. S116-S117 Piñar-Gutiérrez, A., P. Remón-Ruiz, and A. Soto-Moreno, CASE REPORT: Pituitary apoplexy after COVID-19 vaccination. Medicina clinica, 2021. Presentation: No date and time listed

RF25 | PMON50 Insight into Integrated Control of Pituitary Function Revealed Through Analysis of Musashi Target mRNAs

Abstract The anterior pituitary controls growth, metabolism, reproduction and stress responses through the synthesis and secretion of specific hormones. Distinct cell lineages within the anterior pituitary generate specific hormones. Interestingly, the anterior pituitary exhibits a high level of adult cell plasticity that allows committed hormone-producing cells to alter their cell fate and produce different hormones to meet altered organismal demands. The molecular mechanisms underlying this adult cell plasticity have not been fully characterized. Our recent work has implicated the stem cell determinant and sequence-specific mRNA binding protein, Musashi, as a regulator of pituitary cell plasticity. The mechanisms by which Musashi controls pituitary cell plasticity are unknown. In this study, we have sought to identify the mRNA regulatory targets of Musashi in the adult mouse pituitary. Through the use of Musashi-RNA-immunoprecipitation we report a cohort of 1192 pituitary mRNAs that specifically interact with Musashi. mRNAs specific to each of the hormone-producing cell lineages were identified, as well as mRNAs specific to immature stem and progenitor cell lineages. The biological pathways encoded by the Musashi-enriched mRNAs include cell adhesion, cellular homeostasis, unfolded protein responses, protein trafficking, endocrine processes and female pregnancy. Functional analysis of validated mRNAs reveals both activating and inhibitory control of mRNA translation by Musashi. Together, our findings indicate a broad and complex role for Musashi in the post-transcriptional regulation of adult pituitary function. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:58 p.m. - 1:03 p.m.

ODP357 The Effect of Hyperbetacarotenaemia on Pituitary Homeostasis – A Case Series

Abstract Betacarotene is an antioxidant pigment found in red, orange and yellow coloured leafy fruits and vegetables. Recent studies have found that beta carotene is involved in cell cycle arrest with apoptotic effects in ACTH secreting pituitary cells. It may have desirable anti-tumorigenic effects. Hyperbetacarotenaemia is commonly caused by excess intake of carotene rich food. However, correlating the clinical picture with liver, thyroid and lipid profiling, vitamin A concentrations and exclusion of nephrotic syndrome is necessary to underpin the causative mechanism. We present two cases of hyperbetacarotenaemia with clinical manifestations of hypopituitarism. Case 1 – A 55 year old lady presented with partial hypopituitarism. She had a history of restrictive eating disorder and excessive exercise regime. She cycled 140 miles a week and ate 4lbs of leafy vegetables a day. Her pituitary profile reflected partial hypopituitarism with inappropriately undetectable LH/FSG, low IGF-1 and central hypothyroidism. Her cortisol response to tetracosactide testing was normal. Betacarotene reached a peak of 2.7umol/L (upper reference range 1.58umol/L). Case 2 – A 29 year old lady was diagnosed and treated for a 4mm microprolactinoma, which subsequently regressed and prolactin levels returned to normal. Despite this, she continued to experience menstrual irregularities and found to have elevated serum beta carotene levels. On direct questioning she admitted to drinking 1.5L of Lucozade® per day. Betacarotene reached a peak of 3.81umol/L. These cases illustrate the importance of taking a detailed dietary history when considering the causes of pituitary dysfunction. Presentation: No date and time listed

ODP305 Differentiation Potential of Marginal Layer-Derived Adult Pituitary Stem/Progenitor Cells into Vascular Endothelial Cells and Pericytes in Mice

Abstract The anterior lobe (AL) of the pituitary gland consists five types of hormone-producing cells and fenestrated sinusoids (i. e., vascular endothelial cells and pericytes) that cooperate to maintain the physiological function of the gland. In addition, the population of cells located in pituitary stem/ progenitor cell niche, the marginal layer, which faces the residual lumen of the Rathke's pouch, serve to renew pituitary cells throughout life. We recently established a protocol for isolating marginal layer-derived adult pituitary stem/progenitor cells (MPSCs) by using differences in cell adhesion (1), and then showed differentiation potential into hormone-producing cells by subjecting MPSCs to three-dimensional culture. However, it is unclear whether MPSCs are capable of differentiating into all types of cells that constitute the pituitary gland. In this study, we performed DNA microarray analysis in MPSCs, which were proliferated by two-dimensional (2D) culture, for studying comprehensively gene expression profiles. Next, we investigated what kind of cells MPSCs would differentiate into. Comparing gene expression profiles of the AL and MPSCs, in MPSCs, 1776 genes were increased more than 4-fold and 1715 genes were decreased 4-fold compared to the AL (p<0. 05, respectively). The upregulated genes included pituitary stem/progenitor cell markers (ex. Sox2, Sox9, Klf4, Prrx1), niche markers (ex. Cdh1, Krt8), and cell surface antigens (ex. Cd9, Cd34, Cd44). On the other hand, most of the downregulated genes were differentiation markers (ex. Pou1f1, Gata2, Tbx19, Kdr). Next, immunofluorescent analysis revealed that most of the MPSCs were positive for CD34, the hematopoietic stem cell marker. After further 2D culture of MPSCs for 5-7days, vascular endothelial marker PECAM1-positive cells were detected by immunofluorescent analysis, but pericyte marker CSPG4-positive cell not. When TGFβ was applied to 2D culture of MPSCs, CSPG4-positive cells were detected instead of PECAM1-positive cells. Expression levels of Pecam1and Cspg4 were also examined by qPCR analysis, which corresponded to the results of immunofluorescent analysis. On the other hand, expression levels of anterior hormone genes were almost unchanged. These results have demonstrated that MPSCs could differentiate into vascular endothelial cells and pericytes. The present study suggests that pituitary stem/progenitor cells, which derived from the marginal layer, are a source of not only hormone-producing cells but also cells that constitute blood vessels. Reference: (1) Shintani and Higuchi, Stem Cell Res. 2021 Feb 2;52: 102223. Presentation: No date and time listed

RF13 | PMON142 Long Non-coding RNAs in Pituitary Differentiation and Endocrine Function

Abstract Mammalian genomes contain thousands of genes for long non-coding RNA (lncRNAs) that affect the expression of other genes through diverse mechanisms. The lncRNA transcripts are longer than 200 nucleotides and are often capped, spliced and polyadenylated, but they are not translated into proteins. Nuclear lncRNAs modify chromatin structure and transcription in trans or cis by interacting with the DNA, forming R-loops, and recruiting regulatory proteins. Not much is known about the role of lncRNA in pituitary gland differentiation and function, but there are several examples that provide a strong premise for investigating this class of transcripts more deeply. For example, the lncRNA MEG3 is a tumor suppressor that is silenced in non-functioning pituitary adenomas; and lncRNAs have been implicated in regulating the function of key transcription factors such as POU1F1, PITX1, and PITX2. To develop a research resource for the study of lncRNA, we used cell-type specific cre transgenes to tag individual adult pituitary cell types with fluorescent markers and enriched for thyrotropes, gonadotropes and somatotropes using fluorescence activated cell sorting. We determined the transcriptome of each cell population using RNA sequencing and mined the data for lncRNA. We detected hundreds of lncRNA in adult pituitary cells, and some were cell-type specific, and located in immediate proximity of genes that encode pituitary hormones or lineage-specific transcription factors. We discovered that the lncRNA Nr5a1os is enriched in gonadotropes, while Foxl2os is enriched in gonadotropes and thyrotropes. The lncRNA Platr9 is located on the opposite strand near Cga, which encodes the alpha subunit of thyrotropin and gonadotropin. We also mined transcriptome data from pituitary glands collected at embryonic days 12.5 and 14.5 and identified 351 unique lncRNAs, many of which are exclusively expressed during development. Among these is Six3os1, which is located upstream of Six3, a gene associated with holoprosencephaly and hypopituitarism. The location of many of these lncRNAs suggests that they may have a cis-acting regulatory role in pituitary development or physiological function. This research resource sets the foundation for examining the actions of lncRNAs on expression of nearby genes during development and in response to physiological demand. Future research will elucidate their mechanisms of action, determining whether they recruit regulatory proteins to the chromatin and/or form R-loops in these regions, and how this affects expression of the target gene(s). We expect these studies to further our understanding of pituitary biology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP308 Does This Patient Have Sheehan's Syndrome Due to Thrombosis?

Abstract Background Sheehan syndrome (SS) is the necrosis of the anterior pituitary cells due to ischemia. The most common etiology is the result of postpartum hemorrhage, causing hypovolemia and decreased blood supply to an already enlarged pituitary gland, which occurs during pregnancy. Other less common causes are thrombosis, vasospasm, or compression on hypophysial arteries. Clinical Case A 35-year-old lady G1P1 was referred to Endocrinology Clinic for evaluation of secondary amenorrhea and infertility, and a desire for a second child. Prior to birth of her daughter, she had irregular menses every 30-90 days. She did not have excessive bleeding with delivery. However, she developed postpartum deep vein thrombosis, and was treated with enoxaparin for 1 year after delivery. Although she had no symptoms of hypopituitarism, she has not had a menstrual period since delivery of her child and does not exercise vigorously. She did not have symptoms or signs of PCOS. Upon evaluation, Factor V Leiden showed a heterozygous mutation, Estradiol14.98L[19.5-144.2 pg/mL], LH 2.3[ 1.9 - 26.2 mIU/mL], FSH 7.4[2.3 - 12.6 mIU/mL], FT4 1.3 [0.76 - 1.46 ng/dL],TSH 1.10[ 0.358 - 3.74uIU/mL], IGFI 182[ 53 - 331ng/mL], ACTH 12[ 0–47 pg/mL], am Cortisol 15.2[5.3 - 22.5 ug/dL],Testosterone18 [ 2–45 ng/dL],DHEA Sulfate58[35-430mcg/dL] Prolactin 5.7[<25 ng/ml],with normal fallopian tubes on HSG. Pituitary MRI was done due to low estradiol with inappropriately normal FSH and LH and was normal. The patient was diagnosed with Hypogonadotropic deficiency leading to secondary amenorrhea, of unclear etiology, in the setting of possible hereditary caused thrombosis. The concern for hormonal therapy for a second pregnancy was whether this is safe and effective, given her history of DVT and factor V Leiden mutation. Discussion and Conclusion Previous studies have found that 82% of cases with SS were due to postpartum hemorrhage, but also suggests that there are other etiologies for SS. In addition, failure of lactation occurs in 67% of patients and secondary amenorrhea in 73% of patients, as in our case. However, the extent of pituitary involvement can vary, with abnormal GH and prolactin in 90-100% of cases, while abnormal TSH, Cortisol and GnRH in 50-100%. Although anti-phospholipid syndrome has been described as a possible etiology for hypopituitarism, related to formation of thrombi, our case suggests that other coagulopathies should be considered as a cause of partial pituitary dysfunction leading to hypogonadotropic hypogonadism, due to thrombus formation. Presentation: No date and time listed

ODP343 Phoenixin as a Novel Autocrine/Paracrine Factor Stimulating Prolactin Secretion and Gene Expression at the Pituitary Level in Fish Model

Abstract Phoenixin (PNX), a new peptide first identified by bioinformatics, is widely expressed at tissue level and confirmed to be the ligand for the orphan receptor GPR173. Its mature peptide PNX 20 is highly conserved across species and emerged as a novel regulator for reproductive, appetite control, memory retention, plain perception and cardiovascular protection. However, not much is known for the comparative aspects of PNX and the post-receptor signaling mediating PNX action is still unclear. Using grass carp as a model for bony fish/teleost, carp PNX and its receptor GPR173 were cloned and confirmed to be single-copy genes in the carp genome. Using RT-PCR, transcript signals of PNX and GPR173 were found to be widely expressed in different tissues, including the brain-pituitary axis. As revealed by LC/MS/MS and ICC staining, protein signals of PNX and GPR173 could be detected in the pituitary and co-localized in immuno-identified lactotrophs. In primary culture of carp pituitary cells, static incubation with the synthetic peptide of carp PNX 20 was effective in increasing prolactin (PRL) release, PRL cell content and total production of PRL with a parallel rise in PRL mRNA level. The opposite was true for PRL secretion and transcript expression in parallel experiment with removal of endogenous PNX by immunoneutralization using PNX antiserum. Besides the stimulation on PRL signals, similar treatment with PNX 20 could also elevate cAMP and intracellular Ca 2+ levels with rapid phosphorylation of PI3K, Akt, MEK 1/2 and ERK 1/2 in carp pituitary cells. In the same model, except for a lack of effect with PKC blockade, co-treatment with the pharmacological inhibitors for different signalling components of the cAMP/PKA, PLC/IP 3, PI3K/Akt and MAPK cascades was effective in reducing/causing a total blockade of PNX 20 -induced PRL release and gene expression. Our results, as a whole, suggest that (i) PNX produced locally within the carp pituitary, presumably via activation of GPR173, could act as an autocrine/paracrine signal to stimulate PRL secretion and gene expression in carp lactotrophs, and (ii) the stimulatory effects on PRL regulation caused by the local actions of PNX probably are mediated by the cAMP/PKA-, PLC/IP 3 -, PI3K/Akt- and MAPK-dependent signaling mechanisms. Presentation: No date and time listed

Peripheral glucocorticoid receptor antagonism by relacorilant with modest HPA axis disinhibition.

Glucocorticoid stress hormones are produced in response to hypothalamic-pituitary-adrenal (HPA) axis activation. Glucocorticoids are essential for physiology and exert numerous actions via binding to the glucocorticoid receptor (GR). Relacorilant is a highly selective GR antagonist currently undergoing a phase 3 clinical evaluation for the treatment of endogenous Cushing's syndrome. It was found that increases in serum adrenocorticotropic hormone (ACTH) and cortisol concentrations after relacorilant treatment were substantially less than the increases typically observed with mifepristone, but it is unclear what underlies these differences. In this study, we set out to further preclinically characterize relacorilant in comparison to the classical but non-selective GR antagonist mifepristone. In human HEK-293 cells, relacorilant potently antagonized dexamethasone- and cortisol-induced GR signaling, and in human peripheral blood mononuclear cells, relacorilant largely prevented the anti-inflammatory effects of dexamethasone. In mice, relacorilant treatment prevented hyperinsulinemia and immunosuppression caused by increased corticosterone exposure. Relacorilant treatment reduced the expression of classical GR target genes in peripheral tissues but not in the brain. In mice, relacorilant induced a modest disinhibition of the HPA axis as compared to mifepristone. In line with this, in mouse pituitary cells, relacorilant was generally less potent than mifepristone in regulating Pomc mRNA and ACTH release. This contrast between relacorilant and mifepristone is possibly due to the distinct transcriptional coregulator recruitment by the GR. In conclusion, relacorilant is thus an efficacious peripheral GR antagonist in mice with only modest disinhibition of the HPA axis, and the distinct properties of relacorilant endorse the potential of selective GR antagonist treatment for endogenous Cushing's syndrome.

Contribution of PROP1 in the pathogenesis of Cushing’s disease: A preliminary study

Prophet of Pit1 (PROP1) is the pituitary specific transcription factor which was cloned as an upstream regulator of PIT1. Cushing’s disease is caused by corticotroph adenomas which does not belong to the PIT1 lineage. The aim of this study was to elucidate the involvement of PROP1 in the pathogenesis of Cushing’s disease. Since it is difficult to accurately evaluate PROP1 expression in pituitary adenomas by RT-PCR analysis due to the inevitable contamination of surgical tissue specimens with normal pituitary cells, we examined six corticotroph adenomas by immunohistochemistry. Adenomas were classified into 2 subtypes based on their staining patterns of CAM5.2: diffuse and non-diffuse types. It is likely that diffuse-type adenomas are composed of more differentiated tumor cells. PROP1 was positive in all three cases of non-diffuse-type adenomas, but negative in all three cases of diffuse-type adenomas, suggesting that PROP1 was prominently expressed in less differentiated non-PIT1 lineage corticotroph adenomas. This study is the first report to analyze PROP1 expression in pituitary adenomas by immunohistochemistry. PROP1 may have a role in tumorigenesis of pituitary adenomas in Cushing’s disease, and is expected to be a new therapeutic target for refractory pituitary adenomas.

BRF1-mediated paracrine signalling from pituitary stem cells is required for terminal differentiation of pituitary committed progenitors

BRF1-mediated paracrine signalling from pituitary stem cells is required for terminal differentiation of pituitary committed progenitors