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Context Preclinical studies show seliciclib (R-roscovitine) suppresses neoplastic corticotroph proliferation and pituitary adrenocorticotrophic hormone (ACTH) production. Objective To evaluate seliciclib as an effective pituitary-targeting treatment for patients with Cushing disease (CD). Methods Two prospective, open-label, phase 2 trials, conducted at a tertiary referral pituitary center, included adult patients with de novo, persistent, or recurrent CD who received oral seliciclib 400 mg twice daily for 4 consecutive days each week for 4 weeks. The primary endpoint in the proof-of-concept single-center study was normalization of 24-hour urinary free cortisol (UFC; ≤ 50 µg/24 hours) at study end; in the pilot multicenter study, primary endpoint was UFC normalization or ≥ 50% reduction in UFC from baseline to study end. Results Sixteen patients were consented and 9 were treated. Mean UFC decreased by 42%, from 226.4 ± 140.3 µg/24 hours at baseline to 131.3 ± 114.3 µg/24 hours by study end. Longitudinal model showed significant UFC reductions from baseline to each treatment week. Three patients achieved ≥ 50% UFC reduction (range, 55%-75%), and 2 patients exhibited 48% reduction; none achieved UFC normalization. Plasma ACTH decreased by 19% (P = 0.01) in patients who achieved ≥ 48% UFC reduction. Three patients developed grade ≤ 2 elevated liver enzymes, anemia, and/or elevated creatinine, which resolved with dose interruption/reduction. Two patients developed grade 4 liver-related serious adverse events that resolved within 4 weeks of seliciclib discontinuation. Conclusion Seliciclib may directly target pituitary corticotrophs in CD and reverse hypercortisolism. Potential liver toxicity of seliciclib resolves with treatment withdrawal. The lowest effective dose requires further determination.

ContextPostoperative hyponatremia leads to prolonged hospital length of stay and readmission within 30 days.ObjectiveTo assess 3 strategies for reducing rates of postoperative hyponatremia and analyze risk factors for hyponatremia.DesignTwo retrospective analyses and 1 prospective study.SettingTertiary referral hospital.PatientsPatients undergoing transsphenoidal surgery for pituitary adenomas and other sellar and parasellar pathologies.Intervention(s)Phase 1: no intervention. Phase 2: postoperative day (POD) 7 sodium testing and patient education. Phase 3: fluid restriction to 1 L/day on discharge in addition to phase 2 interventions.Main outcome measuresRates of early and delayed hyponatremia and readmissions. Secondary outcomes were risk factors for hyponatremia and readmission costs.ResultsIn phase 1, 296 patients underwent transsphenoidal surgery. Twenty percent developed early and 28% delayed hyponatremia. Thirty-eight percent underwent POD 7 sodium testing. Readmission rates were 15% overall and 4.3% for hyponatremia. In phase 2 (n = 316), 22% developed early and 25% delayed hyponatremia. Eighty-nine percent complied with POD 7 sodium testing. Readmissions were unchanged although severity of hyponatremia was reduced by 60%. In phase 3 (n = 110), delayed hyponatremia was reduced 2-fold [12.7%, relative risk (RR) = 0.52] and readmissions 3-fold [4.6%, RR = 0.30 (0.12–0.73)]; readmissions for hyponatremia were markedly reduced. Hyponatremia readmission increased costs by 30%.ConclusionsRestricting fluid to 1 L/day on discharge decreases rates of delayed hyponatremia and readmissions by 50%. Standardized patient education and POD 7 sodium testing decreases severity of hyponatremia but does not impact readmission rates. These protocols should be considered standard practice for patients undergoing transsphenoidal surgery.

Rationale: Hyponatremia occurs in approximately 20% of patients after transsphenoidal pituitary surgery (TSS). Symptomatic delayed hyponatremia from SIADH is a major cause of morbidity and hospital readmission. Prophylactic fluid restriction (FR) protocols starting 2-5 days after surgery may potentially reduce rates of symptomatic hyponatremia and readmissions. Methods Patients who underwent TSS for sellar and parasellar masses at a tertiary referral center were discharged with a one-liter fluid restriction protocol. Serum sodium levels were measured on post-operative day (POD) 7 with telephone follow-up care. Rates of hyponatremia and readmissions were compared to a cohort of 316 post-operative patients treated prior to the protocol. Numerical variables were summarized as mean ± SD and significance testing calculated by Chi-square and t-test. Results 105 patients were enrolled on the FR protocol upon discharge. Twelve patients were excluded as they had diabetes insipidus or prolonged hospitalization beyond POD 7. Ninety-three patients were included in the analysis, of whom 74.3% had pituitary adenomas, 5.4% Rathke's Cleft cysts, 8.6% meningiomas, 5.4% apoplexy, 2.2% craniopharyngiomas, and 4.3% other masses. Hyponatremia occurred in 13/93 (14%) patients. Mean POD 7 sodium was 137.9 mmol/L across the entire cohort. Two patients (2.1%) were readmitted for hyponatremia, while 3 were re-admitted for other causes. 88/93 (94.6%) of patients complied with the FR protocol. Of the 5 non-compliant patients, 2 developed hyponatremias (40%) compared to 3/88 (3.4%) in those who complied (p=0.008). Mean POD7 sodium was 138 (+/-4.5) mmol/L in those that complied and 135 (+/- 4.7) mmol/L in those who did not. Overall readmission rate for those with postoperative hyponatremia was 23% compared to 2.5% for those with normal sodium levels (p=0.0023). 15.4% of patients with hyponatremia were re-admitted for hyponatremia treatment, compared to 0/80 (0%) of those who did not develop post-operative hyponatremia (p=0.0004). Age and BMI did not impact hyponatremia rates. In 316 patients treated in 2012-2018 prior to the FR protocol, 78 (24.7%) developed delayed hyponatremia with 6% readmitted for hyponatremia. Thirty nine percent of patients with delayed hyponatremia were re-admitted, compared to 7.6% for those without hyponatremia. Compared to patients not on FR, patients on the FR protocol had 50% reduced risk of hyponatremia (OR=0.49 (95% CI 0.26-0.94, p=0.03), and a 3-fold reduced risk of overall readmissions (OR=0.31; 95% CI 0.12-0.81, p=0.0157) and readmission for hyponatremia (OR= 0.34;95% CI 0.08-1.5, p=0.16). A post-operative CSF leak or use of a lumbar drain were associated with an increased readmission rate in the historical cohort. Conclusion Instituting a one-liter daily FR protocol in patients after TSS results in significantly reduced rates of hyponatremia, overall readmissions, and readmission for symptomatic hyponatremia. A FR protocol should become routine practice in the post-operative care of patients undergoing pituitary surgery. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Somatostatin and dopamine receptors are expressed in normal and tumoral somatotroph cells. Upon receptor stimulation, somatostatin and the somatostatin receptor ligands octreotide, lanreotide, and pasireotide, and to a lesser extent, dopamine and the dopamine analogs bromocriptine and cabergoline, suppress growth hormone (GH) secretion from a GH-secreting pituitary somatotroph adenoma. Somatostatin and dopamine receptors are Gαi-protein coupled that inhibit adenylate cyclase activity and cAMP production and reduce intracellular calcium concentration and calcium flux oscillations. Although their main action on somatotroph cells is acute inhibition of GH secretion, they also may inhibit GH production and possibly somatotroph proliferation. These receptors have been reported to create complexes that exhibit functions distinct from that of receptor monomers. Somatostatin suppression of GH is mediated mainly by somatostatin receptor subtype 2 and to a lesser extent by SST5. Human somatostatin receptor subtype 5 has also been shown to harbor mutations associated with GH levels, somatotroph tumor behavior, and somatostatin receptor ligand (SRL) responsiveness. Reviewing current knowledge of somatostatin and dopamine receptor expression and signaling in normal and tumoral somatotroph cells offers insights into mechanisms underlying SRL and dopamine agonist effectiveness in patients with acromegaly.

Cushing’s syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing’s syndrome). Hypercortisolemic features of ectopic Cushing’s syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing’s syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC. We identify an E2F1 cluster binding to the proximal hPOMC promoter region (−42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing’s cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing’s syndrome.

Cushing disease, due to pituitary corticotroph tumor ACTH hypersecretion, drives excess adrenal cortisol production with adverse morbidity and mortality. Loss of glucocorticoid negative feedback on the hypothalamic-pituitary-adrenal axis leads to autonomous transcription of the corticotroph precursor hormone proopiomelanocortin (POMC), consequent ACTH overproduction, and adrenal hypercortisolism. We previously reported that R-roscovitine (CYC202, seliciclib), a 2,6,9-trisubstituted purine analog, suppresses cyclin-dependent-kinase 2/cyclin E and inhibits ACTH in mice and zebrafish. We hypothesized that intrapituitary cyclin E signaling regulates corticotroph tumor POMC transcription independently of cell cycle progression. The aim was to investigate whether R-roscovitine inhibits human ACTH in corticotroph tumors by targeting the cyclin-dependent kinase 2/cyclin E signaling pathway.Primary cell cultures of surgically resected human corticotroph tumors were treated with or without R-roscovitine, ACTH measured by RIA and quantitative PCR, and/or Western blot analysis performed to investigate ACTH and lineage-specific transcription factors. Cyclin E and E2F transcription factor 1 (E2F1) small interfering RNA (siRNA) transfection was performed in murine corticotroph tumor AtT20 cells to elucidate mechanisms for drug action. POMC gene promoter activity in response to R-roscovitine treatment was analyzed using luciferase reporter and chromatin immunoprecipitation assays.R-roscovitine inhibits human corticotroph tumor POMC and Tpit/Tbx19 transcription with decreased ACTH expression. Cyclin E and E2F1 exhibit reciprocal positive regulation in corticotroph tumors. R-roscovitine disrupts E2F1 binding to the POMC gene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways.R-roscovitine inhibits human pituitary corticotroph tumor ACTH by targeting the cyclin E/E2F1 pathway. Pituitary cyclin E/E2F1 signaling is a previously unappreciated molecular mechanism underlying neuroendocrine regulation of the hypothalamic-pituitary-adrenal axis, providing a subcellular therapeutic target for small molecule cyclin-dependent kinase 2 inhibitors of pituitary ACTH-dependent hypercortisolism, ie, Cushing disease.

Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2WT cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2DRY stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR2DRY did not exhibit an enhanced octreotide response as did GpSSTR2WT cells; however, both SSTR2WT-enhanced yellow fluorescent protein (eYFP) and SSTR2DRY-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2WT cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2DRY cells. In vivo SAHA also increased GH release from GpSSTR2WT but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2WT compared with that of either GpSSTR2DRY or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

Cushing disease caused by adrenocorticotropin (ACTH)-secreting pituitary adenomas leads to hypercortisolemia predisposing to diabetes, hypertension, osteoporosis, central obesity, cardiovascular morbidity, and increased mortality. There is no effective pituitary targeted pharmacotherapy for Cushing disease. Here, we generated germline transgenic zebrafish with overexpression of pituitary tumor transforming gene ( PTTG/securin ) targeted to the adenohypophyseal proopiomelanocortin (POMC) lineage, which recapitulated early features pathognomonic of corticotroph adenomas, including corticotroph expansion and partial glucocorticoid resistance. Adult Tg:Pomc-Pttg fish develop neoplastic coticotrophs and pituitary cyclin E up-regulation, as well as metabolic disturbances mimicking hypercortisolism caused by Cushing disease. Early development of corticotroph pathologies in Tg:Pomc-Pttg embryos facilitated drug testing in vivo. We identified a pharmacologic CDK2/cyclin E inhibitor, R-roscovitine (seliciclib; CYC202), which specifically reversed corticotroph expansion in live Tg:Pomc-Pttg embryos. We further validated that orally administered R-roscovitine suppresses ACTH and corticosterone levels, and also restrained tumor growth in a mouse model of ACTH-secreting pituitary adenomas. Molecular analyses in vitro and in vivo showed that R-roscovitine suppresses ACTH expression, induces corticotroph tumor cell senescence and cell cycle exit by up-regulating p27, p21 and p57, and downregulates cyclin E expression. The results suggest that use of selective CDK inhibitors could effectively target corticotroph tumor growth and hormone secretion.

Pituitary targeted pharmacotherapy for Cushing's disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing's disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known.We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2- and SST5-selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2-overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14- and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression.The results indicate that SST5 determines short- and long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible.

The anterior pituitary gland (adenohypophysis) comprises anterior and intermediate lobes (the pars distalis and pars intermedia) arising from placodal ectoderm at the anterior neural ridge. Signaling molecules including SHH, FGF, WNT, BMP and Notch are involved in regulating primordial pituitary proliferation and lineage determination. However, morphogenic events and molecular mechanisms governing anterior and intermediate lobe specification are not clear. Pituitary expression of proopiomelanocortin (POMC), the common precursor for adrenocorticotropin (ACTH) of pars distalis corticotropes and α-melanocyte-stimulating hormone (α-MSH) of pars intermedia melanotropes, provides a unique marker for anterior and intermediate lobe morphogenesis. We performed time-lapse confocal microscopy lineage tracing in live zebrafish embryos expressing GFP driven by the pomc promoter and show distinct migration pathways of POMC cells destined to the anterior and intermediate lobes. Using morpholino oligonucleotides, we show that hypomorphic FGF3 down-regulation induces specific defects of pars intermedia POMC cells while pomc, growth hormone and prolactin expression remain intact in the pars distalis. This lineage-specific process is independent of the FGF3 effect on early pituitary specifying transcription factors as indicated by normal Lim3 and Pit1 expression in hypomorphic FGF3 morphants. These findings suggest that the FGF3 signal, in addition to its previously described role of regulating progenitor proliferation and survival, delineates the melanotrope and corticotrope lineage boundary, contributing to establishment of the pituitary pars distalis and pars intermedia.

Androgen excess is a central feature of polycystic ovary syndrome (PCOS), which affects 6% to 10% of young women. Mammals exposed to elevated androgens in utero develop PCOS-like phenotypes in adulthood, suggesting fetal origins of PCOS. We hypothesize that excess androgen exposure during early embryonic development may disturb the epigenome and disrupt metabolism in exposed and unexposed subsequent generations. Zebrafish were used to study the underlying mechanism of fetal origins. Embryos were exposed to androgens (testosterone and dihydrotestosterone) early at 26 to 56 hours post fertilization or late at 21 to 28 days post fertilization. Exposed zebrafish (F0) were grown to adults and crossed to generate unexposed offspring (F1). For both generations, global DNA methylation levels were examined in ovaries using a luminometric methylation assay, and fasting and postprandial blood glucose levels were measured. We found that early but not late androgen exposure induced changes in global methylation and glucose homeostasis in both generations. In general, F0 adult zebrafish exhibited altered global methylation levels in the ovary; F1 zebrafish had global hypomethylation. Fasting blood glucose levels were decreased in F0 but increased in F1; postprandial glucose levels were elevated in both F0 and F1. This androgenized zebrafish study suggests that transient excess androgen exposure during early development can result in transgenerational alterations in the ovarian epigenome and glucose homeostasis. Current data cannot establish a causal relationship between epigenetic changes and altered glucose homeostasis. Whether transgenerational epigenetic alteration induced by prenatal androgen exposure plays a role in the development of PCOS in humans deserves study.

Diagnosis of adrenal-cortisol insufficiency is often misleading in hospitalized patients, as clinical and biochemical features overlap with comorbidities. We analyzed clinical determinants associated with a biochemical diagnosis of adrenal-cortisol insufficiency in non-intensive care unit (ICU) hospitalized patients.In a retrospective cohort study we reviewed 4668 inpatients with random morning cortisol levels ≤15 μg/dL hospitalized in our center between 2003 and 2010. Using serum cortisol threshold level of 18 μg/dL 30 or 60 minutes after Cortrosyn (250 μg; Amphastar Pharmaceuticals, Inc., Rancho Cucamonga, Calif) injection to define biochemical adrenal-cortisol status, we characterized and compared insufficient (n = 108, serum cortisol ≤18 μg/dL) and sufficient (n = 394; serum cortisol18 μg/dL) non-ICU hospitalized patients.Commonly reported clinical and routine biochemical adrenal-cortisol insufficiency features were similar between insufficient and sufficient inpatients. Biochemical adrenal-cortisol insufficiency was associated with increased frequency of liver disease, specifically hepatitis C (P = .01) and prior orthotopic liver transplantation (P.001), human immunodeficiency virus (HIV; P = .005), and reported pre-existing male hypogonadism (P.001), as compared with the biochemical adrenal-cortisol sufficiency group. Forty percent of insufficient inpatients were not treated with glucocorticoids after diagnosis. Multivariable logistic analysis demonstrated that inpatients with higher cortisol levels (P = .0001) and higher diastolic blood pressure (P = .05), and females (P = .009) were more likely not to be treated, while those with previous short-term glucocorticoid treatment (P = .002), other coexisting endocrine diseases (P = .005), or who received an in-hospital endocrinology consultation (P.0001), were more likely to be replaced with glucocorticoids.Commonly reported adrenal-cortisol insufficiency features do not reliably identify hospitalized patients biochemically confirmed to have this disorder. Comorbidities including hepatitis C, prior orthotopic liver transplantation, HIV, and reported pre-existing male hypogonadism may help identify hospitalized non-ICU patients for more rigorous adrenal insufficiency assessment.

The proximal molecular pathogenesis of ACTH-secreting pituitary ­adenomas remains enigmatic. Several transgenic mice models have contributed important knowledge to understanding human pituitary disease; animal and cell models have provided novel insights into mechanisms underlying the pathogenesis of ACTH-secreting pituitary adenomas, mostly due to cell cycle disruption. Defective glucocorticoid feedback mechanisms also likely lead to enhanced POMC expression and corticotroph proliferation. Novel peptide therapies targeting ­somatostatin and/or dopamine (D2) receptors may also provide further insights into ACTH-secreting pituitary tumor pathogenesis. Studies investigating microRNA expression in pituitary corticotroph adenomas point to important functions of a unique class of gene regulators in the molecular biology of Cushing’s disease. Continuing research advancement will lead to better understanding of Cushing’s disease and development of novel therapeutic approaches.

The matrix metalloproteinase (MMP) family of proteins degrades extracellular matrix (ECM) components as well as processes cytokines and growth factors. MMPs are involved in regulating ECM homeostasis in both normal physiology and disease pathophysiology. Here we report the critical roles of mmp23b in normal zebrafish liver development. Mmp23b was initially identified as a gene linked to the genomic locus of an enhancer trap transgenic zebrafish line in which green fluorescent protein (GFP) expression was restricted to the developing liver. Follow-up analysis of mmp23b messenger RNA (mRNA) expression confirmed its liver-specific expression pattern. Morpholino knockdown of mmp23b resulted in defective hepatocyte proliferation, causing a reduction in liver size while maintaining relatively normal pancreas and gut development. Genetically, we showed that mmp23b functions through the tumor necrosis factor (TNF) signaling pathway. Antisense knockdown of tnfa or tnfb in zebrafish caused similar reductions of liver size, whereas overexpression of tnfa or tnfb rescued liver defects in mmp23b morphants but not vice versa. Biochemically, MMP23B, the human ortholog of Mmp23b, directly interacts with TNF and mediates its release from the cell membrane in a cell culture system. Because mmp23b/MMP23B is highly conserved, our findings in zebrafish warrant further investigation of its role in regulating liver development in mammals.

Subcellular molecular mechanisms underlying ACTH-secreting pituitary adenomas remain elusive. Genetic and acquired animal and cell models have provided insights into corticotroph cell tumorigenesis, including cell cycle abnormalities and aberrant glucocorticoid feedback mechanisms. Novel peptide therapies targeting somatostatin and/or dopamine (D2) receptors and emerging microRNA studies may shed light on additional cellular pathways that regulate tumoral corticotroph cell function.

Somatostatin (SRIF) binds G protein-coupled SRIF receptor subtypes (SST1, -2, -3, -4, and -5) to regulate cell secretion and proliferation. Hypothalamic SRIF inhibits pituitary growth hormone, thyroid stimulating hormone, and ACTH secretion. We tested SRIF-independent constitutive SST activity in AtT20 mouse pituitary corticotroph cells in which ACTH secretion is highly sensitive to SRIF action. Stable transfectants expressing SST2 or SST5 were sensitized to selective agonist action, and constitutive SST receptor activity was demonstrated by forskolin and pertussis toxin cAMP cell responses. Persistent constitutive SST activity decreased cell ACTH responses to CRH through decreased expression of CRH receptor subtype 1. Decreased dopamine receptor type 1 expression was associated with attenuated dopamine agonist action, whereas responses to isoproterenol were enhanced through increased β2-adrenoreceptor expression. Thus, integrated pituitary cell ACTH regulation is determined both by phasic SRIF action, as well as by tonic constitutive SST activity, independently of SRIF.

Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21Cip, p27Kip, and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis., Author Summary The pancreas is a vital organ comprising endocrine and exocrine components. Both endocrine and exocrine cells derive from a common pool of progenitors present in the gut endoderm during embryogenesis. The molecular mechanisms regulating cell fate decisions and lineage-specific proliferation are not fully understood. In this work, we report the characterization of a novel gene, exocrine differentiation and proliferation factor (exdpf), as a regulator for exocrine cell fate and differentiation/proliferation. We show that it is a direct target of the transcription factor pancreas-specific transcription factor 1a (Ptf1a), which is expressed in progenitors that give rise to all pancreatic cell types. We find that a deficiency of exdpf results in a severe reduction of exocrine size due to defects in cell proliferation. Consistent with this finding, overexpression of exdpf leads to an increase of exocrine size and a decrease of endocrine size, suggesting a possible change in fate of the endocrine progenitors. The human ortholog of exdpf is highly conserved and its expression level appears elevated in several cancers, including hepatic and pancreatic cancers, implying a possible role in pathogenesis of these malignancies., The zebrafishexdpf, a novel regulator of pancreatic exocrine cell fate, is essential for exocrine cell differentiation and proliferation.

Anterior pituitary hormone secretion is under tonic suppression by hypothalamic somatostatin signaling through somatostatin receptor subtypes (SSTs). Because some hormonal axes are known to be abnormally regulated by ligand-independent constitutively active G protein-coupled receptors, we tested pituitary SSTs for selective constitutive signaling. We therefore differentially silenced endogenous SST2, SST3, and SST5 in somatostatin-sensitive ACTH-secreting mouse AtT-20 pituitary corticotroph cells using small inhibitory RNA (siRNA) and analyzed downstream SSTs-regulated pathways. Transfection with siRNA reduced specific receptor subtype mRNA expression up to 82%. Specificity of receptor silencing was validated against negative controls with different gene-selective siRNAs, concordance of mRNA and cAMP changes, reduced potency of receptor-selective agonists, and phenotype rescue by overexpression of the silenced receptor. Mouse SST3 > SST5 > SST2 knockdown increased basal cAMP accumulation (up to 200%) and ACTH secretion (up to 60%). SST2- and SST5-selective agonist potencies were reduced by SST3- and SST5-silencing, respectively. SST5 > SST2 = SST3 silencing also increased basal levels of ERK1/2 phosphorylation. SST3- and SST5-knockdown increased cAMP was only partially blocked by pertussis toxin. The results show that SST2, SST3, and SST5 exhibit constitutive activity in mouse pituitary corticotroph cells, restraining adenylate cyclase and MAPK activation and ACTH secretion. SST3 mainly inhibits cAMP accumulation and ACTH secretion, whereas SST5 predominantly suppresses MAPK pathway activation. Therefore, SST receptor subtypes control pituitary cell function not only through somatostatin binding to variably expressed cell membrane receptor subtypes, but also by differential ligand-independent receptor-selective constitutive action.

Bmp, Fgf, and retinoic acid (RA) signals have been implicated as regulators of pancreas development. However, the integration of these signaling pathways in vivo is not fully understood. Variant hnf1 (Vhnf1) is a transcription factor involved in pancreas, liver, and kidney development and its mutation in zebrafish causes underdeveloped pancreas and liver. We investigated the signaling pathways that regulate vhnf1 expression during pancreas development. First, we showed that Bmp activity is required for vhnf1 expression in the endoderm. In chordin (a Bmp antagonist) morpholino (MO)-injected embryos, vhnf1 expression in endoderm and in endocrine β cells is expanded. On the other hand, in alk8 (a type I TGFβ receptor) MO-injected embryos, vhnf1 expression in the endoderm is significantly reduced. Second, we showed that Fgf signaling participates in regulation of pancreas development through the vhnf1 pathway. Third, we demonstrated that RA fails to rescue reduction of insulin expression in vhnf1 mutants, whereas overexpression of vhnf1 restores insulin expression that is repressed by treatment with a RA receptor inhibitor. And finally, we revealed that both Bmp and Fgf signals act genetically upstream of RA in directing pancreas development. Taken together, our data establish that vhnf1 acts downstream of the signaling pathways of RA, Bmp, and Fgf to regulate pancreas development in zebrafish.

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of osmotic homeostasis in the development of PRL-secreting lactotrophs, we generated germline transgenic zebrafish coexpressing red fluorescent protein directed by Prolactin regulatory elements (PRL-RFP) and green fluorescent protein by the Pro-opiomelanocortin promoter (POMC-GFP). Transparent embryos expressing fluorescent markers specifically targeted to lactotrophs and corticotrophs, the two pituitary lineages involved in teleost osmotic adaptation, allowed in vivo dynamic tracing of pituitary ontogeny during altered environmental salinity. Physiological osmotic changes selectively regulate lactotroph but not corticotroph proliferation during early ontogeny. These changes are not suppressed by pharmacological dopamine receptor blockade but are completely abrogated by morpholino knockdown of the PRL receptor. PRL receptor signaling exerts robust effects on lactotroph development and plays a permissive role in lactotroph osmo-responsiveness, reflecting the dual peripheral and central interactions required for early pituitary development and embryonic homeostasis.

We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and αMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10−5m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.

Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis., Author Summary Previous studies have shown that Rb+/− mice develop pituitary adenomas; however, RB1 mutations have not been found in human pituitary tumors. In the present study, we uncovered a novel genetic pathway that may lead to Rb downregulation through RNA splicing mediated by usp39, a gene involved in assembly of the spliceosome. Our forward genetic study in zebrafish suggests that loss of usp39 results in aberrant rb1 mRNA splicing, which likely causes elevated expression of its target e2f4, a key regulator known to have oncogenic activity when overexpressed. We established that e2f4 upregulation is a main factor responsible for the adenohypophyseal cell lineage hyperplasia observed in the zebrafish usp39 mutant. It should be of interest to investigate if mutations or downregulation of USP39 would contribute to pituitary tumorigenesis in humans.

SummaryTransgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.