Nucleobindin-derived nesfatin-1 and nesfatin-1-like peptide stimulate pro-opiomelanocortin synthesis in murine AtT-20 corticotrophs through the cAMP/PKA/CREB signaling pathway.

Nucleobindin (NUCB)-derived peptides, nesfatin-1 (NES-1) and nesfatin-1-like peptide (NLP) have several physiological roles in vertebrates. While NES-1 is implicated in stress, whether NUCB1/NLP and NUCB2/NES-1 have any effect on proopiomelanocortin (POMC) remains unknown. The main aim of this study was to determine if NES-1 and/or NLP affect POMC synthesis in mouse corticotrophs. Immunocytochemistry was employed to target NUCB colocalization with POMC in immortalized mouse tumoral corticotrophs (AtT-20 cells). The ability of NES-1 and NLP to modulate POMC mRNA and protein in AtT-20 cells was assessed by qPCR and Western blot, respectively. Moreover, cell-signaling molecules mediating the effect of NES-1 and NLP on POMC synthesis in mouse tumoral corticotrophs were studied using pharmacological blockers. Mouse tumoral corticotrophs showed immunoreactivity for both NUCB1/NLP and NUCB2/NES-1. Both NES-1 and NLP exerted a stimulatory effect on POMC transcript abundance and protein expression in a dose- and time-dependent manner. This effect was comparable to corticotropin-releasing factor (CRF, positive control) stimulation of POMC. Incubation of mouse tumoral corticotrophs with NES-1 or NLP upregulated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The stimulatory effect of these peptides on POMC transcript abundance and protein expression was blocked by the PKA inhibitor, H89, and an adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA). These pharmacological studies indicate that NES-1 and NLP act through the cAMP/PKA/CREB cellular pathway to stimulate POMC synthesis. Our results provide molecular evidence to support a stimulatory role for nucleobindin-derived peptides on POMC synthesis from corticotrophs. Collectively, this research indicates that corticotrophs produce NUCBs, and the encoded peptides NES-1 and NLP could elicit a direct action to stimulate the pituitary stress hormone. This stimulatory effect is mediated by an uncharacterized G protein-coupled receptor (GPCR) that utilizes the cAMP/PKA/CREB pathway.

Differential effects of ß-arrestin1 and ß-arrestin2 on somatostatin receptors in murine AtT-20 corticotroph tumor cells.

Autonomous production of adrenocorticotropic hormone (ACTH) from pituitary corticotroph adenomas is the primary cause of Cushing's disease. Somatostatin receptor, a G protein-coupled receptor (GPCR), types 2 (SSTR2) and 5 (SSTR5) mRNA expression is greater than that of other SSTR subtypes in human corticotroph adenomas. Further, the multiligand SOM230 shows potent effects in decreasing ACTH plasma levels and urinary free cortisol levels in patients with Cushing's disease. We previously showed that both Sstr2 and Sstr5 mRNA levels were unaffected by SOM230 treatment, suggesting that both receptors might not be downregulated by the agonist. Intracellular molecules, such as ß-arrestins, modulate ligand activated-receptor responses. In the present study, we determined regulation of ß-arrestin1 and ß-arrestin2 by SOM230 and dexamethasone in murine AtT-20 corticotroph tumor cells. In addition, we examined the effects of ß-arrestin1 and ß-arrestin2 on Sstr mRNA and their protein levels. SOM230 treatment increased ß-arrestin1 mRNA levels and did not alter ß-arrestin2 mRNA levels. SOM230 treatment could induce ß-arrestin1 production in corticotroph tumor cells. Dexamethasone treatment decreased ß-arrestin2 mRNA levels. ß-arrestin2 knockdown increased proopiomelanocortin, and both Sstr2 and Sstr5 mRNA and their protein levels. The ß-arrestin2 knockdown-increased proopiomelanocortin mRNA levels were canceled by SOM230 treatment.

Targeting Corticotroph HDAC and PI3-Kinase in Cushing Disease.

CONTEXT: Cushing disease (CD) is a life-threatening disorder. Therapeutic goals include symptom relief, biochemical control, and tumor growth inhibition. Current medical therapies for CD by and large exert no action on tumor growth. OBJECTIVE: To identify drugs that inhibit corticotroph tumor adrenocorticotropic hormone (ACTH) secretion and growth. DESIGN: High throughput screen employing a novel "gain of signal" ACTH AlphaLISA assay. SETTING: Academic medical center. PATIENTS: Corticotroph tumor tissues from patients with CD. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Potent inhibitors of corticotroph tumor ACTH secretion and growth. RESULTS: From a kinase inhibitor library, we identified the dual PI3K/HDAC inhibitor CUDC-907 as a potent inhibitor of murine and human corticotroph tumor ACTH secretion (median effective concentration 1-5 nM), and cell proliferation (median inhibitory concentration 5 nM). In an in vivo murine corticotroph tumor xenograft model, orally administered CUDC-907 (300 mg/kg) reduced corticotroph tumor volume (TV [cm3], control 0.17 ± 0.05 vs CUDC-907 0.07 ± 0.02, P < .05) by 65% and suppressed plasma ACTH (ACTH [pg/mL] control 206 ± 27 vs CUDC-907 47 ± 7, P < .05) and corticosterone (corticosterone [ng/mL] control 180 ± 87 vs CUDC-907 27 ± 5, P < .05) levels by 77% and 85% respectively compared with controls. We also demonstrated that CUDC-907 acts through HDAC1/2 inhibition at the proopiomelanocortin transcriptional level combined with its PI3K-mediated inhibition of corticotroph cell viability to reduce ACTH secretion. CONCLUSIONS: Given its potent efficacy in in vitro and in vivo models of CD, combined with proven safety and tolerance in clinical trials, we propose CUDC-907 may be a promising therapy for CD.

Corticotroph tumor progression during long-term therapy with osilodrostat in a patient with persistent Cushing's disease.

PURPOSE: Corticotroph tumor progression (CTP) or Nelson's syndrome (NS) can occur in patients with Cushing's disease (CD) following bilateral adrenalectomy. It has rarely been observed in patients treated with long-term medical therapy for persistent CD. Osilodrostat (LCI699) is a new steroidogenesis inhibitor of 11ß-hydroxylase (CYP11ß1) that induced remission of hypercortisolism in 86% of patients with refractory CD in the randomized placebo-controlled trial LINC-3 (NCT02180217). METHODS: A 40-year-old woman with persistent CD following transsphenoidal surgery was treated with osilodrostat in the LINC-3 trial and was followed with regular hormonal assessments and imaging of residual corticotroph tumor. RESULTS: Under oral therapy with osilodrostat 10 mg twice daily, urinary free cortisol (UFC) normalized and clinical signs of CD regressed during therapy. However after 4 years of treatment, ACTH levels increased from 73 to 500 pmol/L and corticotroph tumor size increased rapidly from 3 to 14 mm, while UFCs remained well controlled. Surgical resection of an atypical tumor with weak ACTH expression and increased proliferative index (Ki-67 ≥ 8%) resulted in current remission but will require close follow-up. CONCLUSION: This case highlights the importance of monitoring ACTH and corticotroph tumor size in patients with persistent CD, either under effective treatment with steroidogenesis inhibitors or after bilateral adrenalectomy.

Bee Venom Stimulates Hormone Secretion in Rat Somatotroph and Corticotroph Cells: Digital Image Analysis of Secretory Granules.

In this study, we characterized secretory granules in somatotroph (STCs) and corticotroph (CTCs) cells from the anterior pituitary of rats, in conjunction with different experimental treatments with bee venom (BV). In the rats injected for 30 days with daily BV doses equivalent to one sting, we found significant changes in secretory granules' diameter: reduced by 48.15% in STCs and increased by 5.09% in CTCs, and especially a shift to gray into their intensity profile: increased by 237.04% in STCs and by 212.38% in CTCs. In the rats injected with a single high BV dose, the granules' diameter was reduced in both STCs (by 7.14%) and CTCs (by 4.67%-significant) and their gray intensity profile increased by 200% in STCs and by 51.71% in CTCs (both are significant). The changes in the gray profile reflected a reduced content of granules in the cells, consistent with an increase of the plasma levels of GH and ACTH in all cases. We concluded that the reduced hormone cargo of granules in STCs and CTCs resulted from an accelerated cell secretion. The results obtained for the two types of cells correlated, indicating a similar reaction of these secretory cells to the prolonged and acute presence of BV in the organism.

Lapatinib decreases the ACTH production and proliferation of corticotroph tumor cells.

Cushing's disease is almost always caused by hypersecretion of adrenocorticotropic hormone (ACTH) from a pituitary adenoma. A mutation in the deubiquitinase gene USP8 has been found in human ACTH-producing pituitary adenoma cells. This mutational hotspot hyperactivates USP8, rescuing epidermal growth factor receptor (EGFR) from lysosomal degradation and ensuring its sustained signaling in Cushing's disease. An EGFR inhibitor would be an effective anti-tumor agent in EGFR-related tumors. We investigated the effect of a potent dual tyrosine kinase inhibitor, lapatinib, on ACTH production and cell proliferation in AtT-20 mouse corticotroph tumor cells. Lapatinib decreased proopiomelanocortin (Pomc) mRNA levels and ACTH levels in AtT-20 cells and also inhibited cell proliferation, induced apoptosis, and decreased pituitary tumor-transforming gene 1 (Pttg1), a hallmark of pituitary tumors, mRNA levels. KSN/Slc nude mice were subcutaneously inoculated with AtT-20 cells. After 1 week, the mice were randomized either to control or lapatinib groups. The inhibitor decreased the tumor weight of AtT-20 allografts in vivo versus control mice. Lapatinib also significantly decreased Pomc and Pttg1 mRNA levels in the tumor and plasma ACTH and corticosterone levels in vivo. Thus, lapatinib decreases the ACTH production and proliferation of corticotroph tumor cells. An EGFR-targeting therapy could be an important treatment for Cushing's disease.

Somatostatin analogs regulate tumor corticotrophs growth by reducing ERK1/2 activity.

Pasireotide has been associated with tumor shrinkage in patients with Cushing's disease subjected to long term treatment. However, to date the implicated molecular mechanisms are poorly elucidated. Here, we tested pasireotide-mediated cytostatic and cytotoxic effects in ACTH-secreting primary tumor cultures and murine corticotroph tumor cell line, AtT-20 cells. We found somatostatin receptor type 5 (SST5) expressed in 17 different ACTH-secreting tumors and SST2 detectable in 15 out of the 17 tissues. Pasireotide caused a slight but significant in vitro inhibition of cell growth in 3 out of 6 ACTH-secreting primary cultures (-12.1 ±â€¯4.3%, P < 0.01 at 10 nM), remarkably reduced phospho-ERK1/2 levels in 5 out of 8 samples (-36.4 ±â€¯20.5%, P < 0.01 at 1 µM) and triggered an increase of caspase 3/7 activity in 2 of 4 tumors (17 ±â€¯3.6%, P < 0.05 at 1 µM). Accordingly, in AtT-20 cells, pasireotide significantly inhibited cell proliferation (-10.5 ±â€¯7.7% at 10 nM, P < 0.05; -13.9 ±â€¯10.9% at 100 nM, P < 0.05; -26.8 ±â€¯8.9% at 1 µM, P < 0.01). Similar antiproliferative actions were exerted by BIM23206 and BIM23120 (SST5&2 selective ligands, respectively), whereas octreotide was effective when used at 1 µM (-13.3 ±â€¯9.1%, P < 0.05). Moreover, a reduction of phospho-ERK1/2 was observed upon pasireotide and BIM23206 treatment (-8.4 ±â€¯28.6%, P < 0.01 and -51.4 ±â€¯15.9%, P < 0.001 at 10 nM, respectively) but not after octreotide and BIM23120 incubation. Finally, pasireotide was able to induce cell apoptosis in AtT-20 cells at lower concentration than octreotide. Altogether these data indicate a downstream implication of SST5-mediated phospho-ERK1/2 inhibition by pasireotide resulting in ACTH-secreting tumor cells proliferation reduction. Moreover, we describe for the first time a pro-apoptotic effect of pasireotide in corticotrophs.

Tumor-Directed Therapeutic Targets in Cushing Disease.

Context: The most frequent cause of endogenous hypercortisolism is Cushing disease (CD), a devastating condition associated with severe comorbidities and high mortality. Effective tumor-targeting therapeutics are limited. Design: Search in PubMed with key words "corticotroph" and "Cushing's disease" plus the name of the mentioned therapeutic agent and in associated references of the obtained papers. Additionally, potential therapeutics were obtained from ClinicalTrials.gov with a search for "Cushing disease." Results: At present, the tumor-targeted pharmacological therapy of CD is concentrated on dopamine agonists (cabergoline) and somatostatin analogs (pasireotide) with varying efficacy, escape from response, and considerable side effects. Preclinical studies on corticotroph pathophysiology have brought forward potential drugs such as retinoic acid, silibinin, and roscovitine, whose efficacy and safety remain to be determined. Conclusions: For many patients with CD, effective tumor-targeted pharmacological therapy is still lacking. Coordinated efforts are pivotal in establishing efficacy and safety of novel therapeutics in this rare but devastating disease.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

Metformin suppresses growth and adrenocorticotrophic hormone secretion in mouse pituitary corticotroph tumor AtT20 cells.

Pituitary corticotroph tumors lead to excess adrenocorticotrophic hormone (ACTH) secretion, resulting in Cushing's disease (CD), which is associated with significant mortality. Standard treatments include neurosurgery, radiotherapy and medical therapy. Both surgery and radiotherapy have undesirable complications and high recurrence rates. At present, there is only one medical option available that targets pituitary adenoma and ACTH secretion, the drug pasireotide. However, hyperglycemia is common during pasireotide treatment. In addition, some patients have discontinued pasireotide treatment because of hyperglycemia-related adverse events or uncontrolled diabetes. New medical treatments directly targeting the corticotroph cells and suppressing ACTH secretion are urgently required. Metformin is a commonly used antidiabetic drug that has been widely used to control the hyperglycemia that occurs in patients with CD, which is secondary to both cortisol excess and pasireotide treatment. Recent studies suggest that metformin has direct anticancer activities against many tumor cell lines. In the present study, we investigated whether metformin exerts an anti-tumor effect by directly targeting pituitary corticotroph tumors and exploring the underlying mechanisms. Using the mouse corticotroph tumor cells, AtT20 cells, we report that metformin inhibited cell proliferation, promoted cell apoptosis and decreased ACTH secretion but did not block the cell cycle in cells. The apoptosis induced by metformin was accompanied by increased caspase-3 activity. Meanwhile, metformin down-regulated the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) but up-regulated the pro-apoptotic protein Bcl2-associated X (BAX), which suggests the involvement of the mitochondrial-mediated apoptosis pathway. Furthermore, metformin promoted AMP-activated protein kinase (AMPK) phosphorylation but inhibited insulin-like growth factor-1 receptor (IGF-1R) expression, protein kinase B (PKB/AKT) phosphorylation and mammalian target of rapamycin (mTOR) phosphorylation. Finally, the IGF-1R inhibitor picropodophyllin (PPP) significantly inhibited the cell proliferation of AtT20 cells. We conclude that metformin inhibits cell proliferation and induces apoptosis in AtT20 cells by activating AMPK/mTOR and inhibiting IGF-1R/AKT/mTOR signaling pathways. Metformin may have direct antitumor activity against pituitary corticotroph tumors.

Role of the ubiquitin/proteasome system on ACTH turnover in rat corticotropes.

PURPOSE: A large number of studies has investigated proopiomelanocortin processing in anterior pituitary corticotropes but little is known on proopiomelanocortin/ACTH degradation within these cells. The ubiquitin-proteasome system is an intracellular protein degradation pathway which has garnered considerable interest in recent times, given its role in maintenance of protein homeostasis. Aim of the present study was to evaluate the role of the ubiquitin-proteasome system in proopiomelanocortin/ACTH turnover in pituitary corticotropes. METHODS: Rat anterior pituitary primary cultures were treated with 0.01-100 nM MG132, a proteasome inhibitor, or 0.1-100 nM K48R, an inhibitor of polyubiquitylation, for 4 and 24 h and ACTH concentrations in medium and cell lysates estimated by immunometric assay. Co-immunoprecipitation for ubiquitin and ACTH was carried out to establish ubiquitin-tagged protein products. RESULTS: Inhibition of proteasome-mediated degradation with MG132 lead to an increase in ACTH concentrations, both as regards secretion and cell content. Likewise, inhibition of polyubiquitylation was associated with increased ACTH secretion and cell content. Ubiquitin/ACTH co-immunoprecipitation revealed that proopiomelanocortin was a target of ubiquitylation. CONCLUSIONS: We provide the first evidence that the ubiquitin-proteasome system is involved in proopiomelanocortin/ACTH degradation in corticotropes. Indeed, proopiomelanocortin is a target of ubiquitylation and modulation of ubiquitin-proteasome system affects ACTH turnover. This study shows that regulation of ACTH proteolytic degradation may represent a means to control ACTH secretion.

Triptolide suppresses growth and hormone secretion in murine pituitary corticotroph tumor cells via NF-kappaB signaling pathway.

Triptolide is a principal diterpene triepoxide from the Chinese medical plant Tripterygium wilfordii Hook. f., whose extracts have been utilized in dealing with diverse diseases in traditional Chinese medicine for centuries. Recently, the antitumor effect of triptolide has been found in several pre-clinical neoplasm models, but its effect on pituitary corticotroph adenomas has not been investigated so far. In this study, we are aiming to figure out the antitumor effect of triptolide and address the underlying molecular mechanism in AtT20 murine corticotroph cell line. Our results demonstrated that triptolide inhibited cell viability and colony number of AtT20 cells in a dose- and time-dependent pattern. Triptolide also suppressed proopiomelanocortin (Pomc) mRNA expression and extracellular adrenocorticotropic hormone (ACTH) secretion in AtT20 cells. Flow cytometry prompted that triptolide leaded to G2/M phase arrest, apoptosis program and mitochondrial membrane depolarization in AtT20 cells. Moreover, dose-dependent activation of caspase-3 and decreased Bcl2/Bax proportion were observed after triptolide treatment. By western blot analysis we found that triptolide impeded phosphorylation of NF-κB p65 subunit and extracellular signal-regulated kinase (ERK), along with reduction of cyclin D1, without any impact on other NF-κB related protein expression like total p65, p50, IκB-α, p-IκB-α. Furthermore, the mouse xenograft model revealed the inhibition of tumor growth and hormone secretion after triptolide administration. Altogether this compound might be a potential pharmaceutical choice in managing Cushing's disease.

Tanshinone IIA induces apoptosis via inhibition of Wnt/ß­catenin/MGMT signaling in AtT­20 cells.

A strategy to suppress the expression of the DNA repair enzyme O6­methylguanine­DNA methyltransferase (MGMT) by inhibition of Wnt/ß­catenin signaling may be useful as a novel treatment for pituitary adenoma. Previous studies have reported that Tanshinone IIA (TSA), a major quinone compound isolated from Salvia miltiorrhiza, had antitumor effects. However, whether TSA has antitumor effects against pituitary adenoma and whether the mechanisms are associated with the Wnt/ß­catenin/MGMT pathway remains to be clarified. In the present study, TSA treatment caused apoptosis in AtT­20 cells in a concentration­dependent manner, as demonstrated by cell viability reduction, phophatidylserine externalization detected by Annexin V staining and mitochondrial membrane potential disruption detected by JC­1 staining, which were associated with activation of caspase­3 and DNA fragmentation detected by TUNEL in AtT­20 cells. T­cell factor (TCF)­lymphoid­enhancing factor (LEF) reporter activity was determined by dual luciferase reporter assay and the interaction between ß­catenin and TCF­4 were detected using a co­immunoprecipitation kit. The results indicated TSA treatment increased ß­catenin phosphorylation, inhibited ß­catenin nuclear translocation, reduced ß­catenin/TCF­4 complex formation and TCF­LEF luciferase reporter activity, and subsequently reduced the expression of cyclin D1 and MGMT. Notably, overexpression of MGMT in ß­catenin knock down AtT­20 cells abrogated the TSA­mediated effects in AtT­20 cells. In conclusion, TSA induced apoptosis via inhibition of Wnt/ß­catenin­dependent MGMT expression, which may provide novel insights into the understanding of the mechanism of the antitumor effects of Salvia miltiorrhiza.

Histone Deacetylase Inhibitor SAHA Is a Promising Treatment of Cushing Disease.

Context: Remission failure following transsphenoidal surgery in Cushing disease (CD) from pituitary corticotroph tumors (CtTs) remains clinically challenging. Histone deacetylase inhibitors (HDACis) are antitumor drugs approved for clinical use, with the potential to affect adrenocorticotropin hormone (ACTH) hypersecretion by inhibiting pro-opiomelanocortin (POMC) transcription. Objective: Testing the efficacy of suberoylanilide hydroxamic acid (SAHA) on human and murine ACTH-secreting tumor (AtT-20) cells. Design: Cell viability, ACTH secretion (enzyme-linked immunosorbent assay), apoptosis, and gene expression profile were investigated on AtT-20 cells. In vivo efficacy was examined in an athymic nude mouse AtT-20 xenograft model. SAHA efficacy against human-derived corticotroph tumor (hCtT) (n = 8) was tested in vitro. Setting: National Institutes of Health. Intervention: SAHA (0.5 to 8 µM). Main Outcome Measures: AtT-20 and hCtT cell survival, in vitro/invivo ACTH measurements. Results: SAHA (1 µM) reduced AtT-20 viability to 75% at 24 hours, 43% at 48 hours (analysis of variance; P = 0.002). Apoptosis was confirmed with elevated BAX/Bcl2 ratio and FACS. Intriguingly, early (3-hour) significant decline (70%; P < 0.0001) of secreted ACTH and diminished POMC transcription was observed with SAHA (1 µM). Microarray analysis revealed a direct association between liver X receptor alpha (LXRα) and POMC expression. Accordingly, SAHA reduced LXRα in AtT-20 cells but not in normal murine corticotrophs. Xenografted nude-mice tumor involution (126 ± 33/160 ± 35 vs 337 ± 49 mm3; P = 0.0005) was observed with 5-day intraperitoneal SAHA, with reversal of elevated ACTH (P < 0.0001). SAHA did not affect serum ACTH in nontumor mice. Lastly, we confirmed that SAHA (1 µM/24 h) decreased hCtT survival (78.92%; P = 0.0007) and ACTH secretion (83.64%; P = 0.03). Conclusion: Our findings demonstrate SAHA's efficacy in reducing survival and ACTH secretion in AtT-20 and hCtT cells, providing a potential intervention for recurrent/unremitting CD.

Heterogeneity of Calcium Responses to Secretagogues in Corticotrophs From Male Rats.

Heterogeneity in homotypic cellular responses is an important feature of many biological systems, and it has been shown to be prominent in most anterior pituitary hormonal cell types. In this study, we analyze heterogeneity in the responses to hypothalamic secretagogues in the corticotroph cell population of adult male rats. Using the genetically encoded calcium indicator GCaMP6s, we determined the intracellular calcium responses of these cells to corticotropin-releasing hormone and arginine-vasopressin. Our experiments revealed marked population heterogeneity in the response to these peptides, in terms of amplitude and dynamics of the responses, as well as the sensitivity to different concentrations and duration of stimuli. However, repeated stimuli to the same cell produced remarkably consistent responses, indicating that these are deterministic on a cell-by-cell level. We also describe similar heterogeneity in the sensitivity of cells to inhibition by corticosterone. In summary, our results highlight a large degree of heterogeneity in the cellular mechanisms that govern corticotroph responses to their physiological stimuli; this could provide a mechanism to extend the dynamic range of the responses at the population level to allow adaptation to different physiological challenges.

Distribution of Corticotrophin-Releasing Factor Type 1 Receptor-Like Immunoreactivity in the Rat Pituitary.

Corticotrophin-releasing factor (CRF) regulates the hypothalamic-pituitary-adrenal axis response to stress through its type 1 receptor (CRF1 ) in the corticotrophs of the anterior pituitary. Although CRF1 mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF1 protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177-188 sequence of the first extracellular loop of the rat CRF1 . Using the antiserum, CRF1 -like immunoreactivity (CRF1 -LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF1 -LI also appeared in the intermediate lobe cells and on the fibre-like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF1 (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF1 -LI-positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF1 -LI-positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF1 in the intermediate lobe (98.9 ± 0.2%). CRF1 -LI and the percentage of CRF1 -LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 µg/kg bw). These results suggest that CRF1 is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF1 are regulated by glucocorticoids in the rat anterior pituitary.

Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP.

Derivation of Diverse Hormone-Releasing Pituitary Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) provide an unlimited cell source for regenerative medicine. Hormone-producing cells are particularly suitable for cell therapy, and hypopituitarism, a defect in pituitary gland function, represents a promising therapeutic target. Previous studies have derived pituitary lineages from mouse and human ESCs using 3D organoid cultures that mimic the complex events underlying pituitary gland development in vivo. Instead of relying on unknown cellular signals, we present a simple and efficient strategy to derive human pituitary lineages from hPSCs using monolayer culture conditions suitable for cell manufacturing. We demonstrate that purified placode cells can be directed into pituitary fates using defined signals. hPSC-derived pituitary cells show basal and stimulus-induced hormone release in vitro and engraftment and hormone release in vivo after transplantation into a murine model of hypopituitarism. This work lays the foundation for future cell therapy applications in patients with hypopituitarism.

Glucocorticoids Inhibit CRH/AVP-Evoked Bursting Activity of Male Murine Anterior Pituitary Corticotrophs.

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which governs the neuroendocrine response to stress. Corticotrophs are electrically excitable and fire spontaneous single-spike action potentials and also display secretagogue-induced bursting behavior. The HPA axis function is dependent on effective negative feedback in which elevated plasma glucocorticoids result in inhibition at the level of both the pituitary and the hypothalamus. In this study, we have used an electrophysiological approach coupled with mathematical modeling to investigate the regulation of spontaneous and CRH/arginine vasopressin-induced activity of corticotrophs by glucocorticoids. We reveal that pretreatment of corticotrophs with 100 nM corticosterone (CORT; 90 and 150 min) reduces spontaneous activity and prevents a transition from spiking to bursting after CRH/arginine vasopressin stimulation. In addition, previous studies have identified a role for large-conductance calcium- and voltage-activated potassium (BK) channels in the generation of secretagogue-induced bursting in corticotrophs. Using the dynamic clamp technique, we demonstrated that CRH-induced bursting can be switched to spiking by subtracting a fast BK current, whereas the addition of a fast BK current can induce bursting in CORT-treated cells. In addition, recordings from BK knockout mice (BK(-/-)) revealed that CORT can also inhibit excitability through BK-independent mechanisms to control spike frequency. Thus, we have established that glucocorticoids can modulate multiple properties of corticotroph electrical excitability through both BK-dependent and BK-independent mechanisms.