Murine Corticotroph Research Articles

CSIG-28. R17 INDUCES A PRO-ONCOGENIC PROTEIN HYPERPHOSPHORYLATION PROGRAM IN CUSHING’S DISEASE

Abstract OBJECTIVES to investigate molecular drivers of CD. METHODS We performed single-nucleus assays for transposase-accessible chromatin (snATACseq) and single-nucleus RNAseq (snRNAseq) in 4 CD adenomas including two adenoma-normal pairs. To assess proteins bound to the R17 promoter, we performed reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin and R17 promoter gRNAs followed by mass spectrometry (LC-MS). Mechanistic studies were performed in murine corticotroph cells (mCort) stably overexpressing R17 versus GFP. Using LC-MS, we quantified total and phospho-peptides from human CD adenomas versus adjacent normal pituitary glands (n = 3/group) and from mCort-R17 versus mCort-GFP cells (n = 5/group). RESULTS We found increase in R17 chromatin accessibility and gene expression in CD versus normal corticotrophs. rChIP identified R17 promoter enrichment of SUB1 and histone variant H1.4 indicating active remodeling. In CD specimens and in mCort-R17 cells, PP2A targets EGFR and ERK were hyperphosphorylated. LC-MS identified 30 peptides differentially phosphorylated in both CD adenomas and mCort-R17 cells. mCort-R17 cells demonstrated accelerated cell cycle progression with increased proliferation (P < 0.001). In mCort-R17 cells, the PP2A agonist Fingolimod partially rescued AKT phosphorylation and induced apoptosis. CONCLUSIONS R17 is activated in CD due to upstream chromatin remodeling events. Aberrant R17 signaling leads to protein hyperphosphorylation and cell cycle acceleration, which is partially reversible by the PP2A agonist Fingolimod. Fingolimod may represent a novel therapeutic option for patients with CD.

OR01-02 Epigenomic Dysregulation Of PPP1R17 Drives The Cell Cycle In Adenomas Causing Cushing’s Disease

Abstract Disclosure: D. Asuzu: None. D. Mullaney: None. S. Stoica: None. D. Mandal: None. D. Maric: None. A. Elkahloun: None. B. Sisay: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Pituitary adenomas causing Cushing’s disease are largely (60%) mutationally bland. Previously, with syngeneic, pairwise transcriptomic analysis, we found a robust upregulation of PPP1R17 in canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ CD adenomas. PPP1R17 is an endogenous inhibitor of tumor suppressor phosphatase PP2A. Here, we investigated the epigenomic dysregulation of PPP1R17, its interaction with PP2A, and downstream effects on cell cycle regulation. Methods: We performed single nucleus assays for transposase accessible chromatin (snATACseq, 10X Genomics) in adenomas from 4 CD patients, and two syngeneic adenoma-normal pairs along with parallel single nucleus RNAseq (snRNAseq). Transcription factors bound to open chromatin were identified by reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin (Millipore Sigma) and promoter-specific synthetic guide RNAs (Invitrogen TrueGuide). Bound transcription factors were quantified using mass spectrometry (SimulTOF 300). Fixed human CD adenomas (n=5) were sectioned and stained using multiplexed immunohistochemistry (mIHC). Association between R17 and protein phosphatase 2A (PP2A) was assessed using proximity ligation assays (PLA, Sigma). We overexpressed R17 or GFP controls in murine corticotroph (mCort) cell lines and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). Results: We mapped the chromatin accessibility landscape of CD adenomas compared to normal corticotrophs. We identified coordinated chromatin accessibility (snATACseq) and transcriptional upregulation (snRNAseq) at the R17 locus exclusively within adenomatous corticotrophs. Using rChIP, we identified enrichment at the R17 promoter of the RNA polymerase II transcriptional coactivator p15 (SUB1) and the histone variant H1.4, indicating active chromatin remodeling. PPP1R17 protein and its target PP2A were overexpressed in the adenoma compartment but not in the adjacent normal gland. PLA confirmed robust interaction between R17 and PP2Ac within the CD adenoma compartment. Mechanistically, mCortR17 cells showed inhibition of PP2A with phosphorylation its known targets - EGFR and ERK. mCortR17 cells showed accelerated cell cycle progression compared to mCortGFP (S-phase 16.4% versus 24.1%, P < 0.001) with increased proliferation (two-way ANOVA P < 0.001). PP2A agonists Fingolimod, DT061 and ABL127 led to proliferation arrest in mCortR17 cells. Conclusions: We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas. We found chromatin accessibility underlying PPP1R17 overexpression. PP2A agonists reversed the effects of PPP1R17 overexpression in-vitro. Our findings uncover a novel therapeutic phosphatase target for patients with CD. Presentation: Thursday, June 15, 2023

THU478 Unchecked Kinase Activation Underlies Pituitary Adenomas Causing Cushing’s Disease

Abstract Disclosure: D. Asuzu: None. D. Mullaney: None. Y. Li: None. D. Mandal: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Common mechanisms underlying canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ pituitary adenomas causing Cushing’s disease remain unknown. Using syngeneic, pairwise transcriptomic analysis at the single cell/nucleus resolution, we found a ubiquitous and robust overexpression of PPP1R17 in CD adenoma corticotrophs. The role of PPP1R17, an endogenous inhibitor of tumor-suppressor phosphatase PP2A in the embryonic context remains unknown in the pituitary. Here, we elucidated mechanistically, the effect of PPP1R17 on the phopshoproteome of CD adenomas. Methods: We quantitatively profiled the proteome and phosphoproteome of three pairs of CD adenomas and their syngeneic adjacent normal pituitary glands with TMT labeling and mass spectrometry (LC-MS/MS). We validated the mechanisms with murine corticotroph cells (mCort) overexpressing R17 (mCortR17) or GFP (mCortGFP). Elevated total and phosphprotein expression was assessed using R packages by performing cross sample normalization, noise filtering and differential protein abundance or phosphorylation analyses. Pathway enrichment analysis was performed on the KEGG database using ShinyGo 0.76.2. Results: We identified 37 proteins differentially phosphorylated in CD adenomas compared to adjacent normal pituitary glands, including the AP2-associated protein kinase 1 (Aak1) and the apoptotic chromatin condensation inducer Acin1. Of these 37 proteins, 30 (81%) were also differentially phosphorylated in mCortR17 versus mCortGFP cells, suggesting that differential phosphorylation in CD is driven by R17 hyperactivity. Of the 35 proteins overabundant in CD adenomas, 15 (43%) overlapped with mCortR17, including the Myc-associated protein (Max) and Presenilin 1 (Psen1). Proteins enriched in both human CD and mCort datasets were involved in NOTCH signaling and neurotransmitter release, while phospho-proteome profiling highlighted R17-mediated perturbation of RNA polymerase II transcription termination and mRNA processing. Conclusions:We first discovered a heavily dysregulated phosphoproteome in CD adenomas compared to syngeneic normal glands. We found that we could replicate the signature phosphoproteomic profile by overexpressing PPP1R17 in-vitro. These data indicate a central role for phosphatase (PP2A) inhibition in CD adenomas, and suggest potential novel anti-tumor targets for patients with CD. Presentation: Thursday, June 15, 2023

519 Single Nucleus ATAC Sequencing Reveals Increased PPP1R17 Chromatin Accessibility Driving Cushing’s Disease

INTRODUCTION: Epigenetic drivers of pituitary adenomas causing Cushing’s disease (CD) are virtually unknown. Here, we investigate the epigenetic mechanisms underlying gene dysregulation in CD. METHODS: We isolated single nuclei from 3 CD adenoma – normal gland pairs and performed single nucleus RNA sequencing (snRNAseq) and assays for transposase-accessible chromatin (snATACseq, 10x Genomics). We integrated these data with prior paired single cell data (scRNAseq, n = 3) and bulk RNAseq (CD = 3, non-CD = 3). We performed bulk DNA methylation assays on independent adenomas (CD = 3, non-CD = 3; Illumina Infinium MethylationEPIC). CD adenomas were examined by multiplexed immunohistochemistry (mIHC). We generated murine corticotroph (mCort) cell lines stably transfected with lentiviral expression vectors (Origene) and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). RESULTS: snATACseq and snRNAseq data identified increased chromatin accessibility with upregulated PPP1R17 expression in single nuclei from CD adenomas, in agreement with our prior scRNAseq and bulk RNAseq data (16-fold in paired analysis, FDR < 0.01). DNA methylation failed to identify global or local differential methylation at the PPP1R17 locus. mIHC confirmed PPP1R17 protein overexpression in human CD tumors. PPP1R17 overexpression in mCort cells accelerated the cell cycle with fewer cells accumulating in S-phase (16.4% vs 24.1%, P < 0.01), and increased cell proliferation (two-way ANOVA P < 0.01). CONCLUSIONS: We identified coordinated chromatin accessibility, transcriptional activation and protein expression of PPP1R17 in human CD adenomas compared to surrounding normal tissues, and recapitulated hyperproliferation in-vitro by stable PPP1R17 overexpression. Our findings uncover targetable epigenetic mechanisms in CD pathogenesis.

The Expression of Cell Cycle-Related Genes in USP8-Mutated Corticotroph Neuroendocrine Pituitary Tumors and Their Possible Role in Cell Cycle-Targeting Treatment.

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes' expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

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

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

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.

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

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. Testing the efficacy of suberoylanilide hydroxamic acid (SAHA) on human and murine ACTH-secreting tumor (AtT-20) cells. 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. National Institutes of Health. SAHA (0.5 to 8 µM). AtT-20 and hCtT cell survival, in vitro/invivo ACTH measurements. 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). 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.

Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis.

Cushing’s disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to suppressive negative feedback by glucocorticoids. The abnormal expression of 11beta-hydroxysteroid dehydrogenase (11HSD), which is a cortisol metabolic enzyme, is found in human and murine corticotroph adenomas. Our recent studies demonstrated that canine corticotroph adenomas also have abnormal expression of 11HSD. 11HSD has two isoforms in dogs, 11HSD type1 (HSD11B1), which converts cortisone into active cortisol, and 11HSD type2 (HSD11B2), which converts cortisol into inactive cortisone. It has been suggested that glucocorticoid resistance in corticotroph tumors is related to the overexpression of HSD11B2. Therefore it was our aim to investigate the effects of carbenoxolone (CBX), an 11HSD inhibitor, on the healthy dog’s pituitary-adrenal axis. Dogs were administered 50 mg/kg of CBX twice each day for 15 days. During CBX administration, no adverse effects were observed in any dogs. The plasma adrenocorticotropic hormone (ACTH), and serum cortisol and cortisone concentrations were significantly lower at day 7 and 15 following corticotropin releasing hormone stimulation. After completion of CBX administration, the HSD11B1 mRNA expression was higher, and HSD11B2 mRNA expression was significantly lower in the pituitaries. Moreover, proopiomelanocortin mRNA expression was lower, and the ratio of ACTH-positive cells in the anterior pituitary was also significantly lower after CBX treatment. In adrenal glands treated with CBX, HSD11B1 and HSD11B2 mRNA expression were both lower compared to normal canine adrenal glands. The results of this study suggested that CBX inhibits ACTH secretion from pituitary due to altered 11HSD expressions, and is potentially useful for the treatment of canine Cushing’s disease.

Control of hypothalamic–pituitary–adrenal stress axis activity by the intermediate conductance calcium‐activated potassium channel, SK4

The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4(-/-)). In addition, Kcnn4(-/-) mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4(-/-) mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.

Opposite effects of serum- and glucocorticoid-regulated kinase-1 and glucocorticoids on POMC transcription and ACTH release

Serum- and glucocorticoid-regulated kinase-1 (SGK1) is a glucocorticoid early-response gene; its function, however, has been elucidated mainly in the context of mineralocorticoid signaling. Here, we investigate the expression and function of SGK1 in the pituitary gland, one of the primary glucocorticoid targets. SGK1 is expressed in the human pituitary gland and colocalizes to ACTH. The AtT-20 murine corticotroph cell line was used for functional experiments. Glucocorticoids upregulated SGK1 mRNA and protein levels, parallel to decreasing proopiomelanocortin (POMC) transcription and ACTH release. Dexamethasone-induced changes in SGK1 protein were abolished by the steroid receptor antagonist RU-486 and reduced by the inhibition of PI 3-kinase with LY-294002. SGK1 overexpression increased CREB- and activator protein-1-dependent transcription, POMC transcription, and ACTH secretion but did not influence intracellular cAMP levels. SGK1 overexpression and corticotropin-releasing hormone had additive effects on POMC promoter activity but not on ACTH secretion. SGK1 knockdown by RNA interference decreased POMC promoter activity, demonstrating the importance of SGK1 for basal POMC signaling. In summary, SGK1 is strongly stimulated by glucocorticoids in pituitary corticotrophs; however, its effects on POMC transcription are antagonistic to the classical inhibitory glucocorticoid action, suggesting a cell-regulated counterregulatory mechanism to potentially detrimental glucocorticoid effects.

CRHR1-dependent effects on protein expression and posttranslational modification in AtT-20 cells

Corticotropin-releasing hormone (CRH) plays a major role in coordinating the organism's stress response, including the activity of the hypothalamic-pituitary-adrenocortical axis. The molecular underpinnings of CRH-dependent signal transduction mechanisms in the anterior pituitary have not yet been revealed in detail. In order to dissect the signal transduction cascades activated by CRH receptor type 1, a comparative proteome approach was performed in vitro utilizing murine corticotroph AtT-20 cells. Alterations in protein expression and posttranslational modification in response to CRH stimulation were studied by 2D gel electrophoresis. Selected candidates were analyzed by immunoblotting and quantitative real-time PCR. The differential analyses revealed proteins regulated or modified related to diverse cellular processes. Amongst others we identified alterations in PRKAR1A, the regulatory subunit of protein kinase A; in PGK1 and PGAM1, key regulators of glycolysis; and in proteins involved in proteasome-mediated proteolysis, PSMC2 and PSMA3. These results offer novel entry points to molecular mechanisms underlying stress responses elicited via the hypothalamic-pituitary-adrenocortical axis.

G Protein-Coupled Receptor Kinase 2 Involvement in Desensitization of Corticotropin-Releasing Factor (CRF) Receptor Type 1 by CRF in Murine Corticotrophs

Hypothalamic CRF stimulates synthesis and secretion of ACTH via CRF receptor type 1 (CRFR1) in the anterior pituitary gland. After agonist-activated stimulation of receptor signaling, CRFR1 is down-regulated and desensitized. Generally, it is thought that G protein-coupled receptors may be desensitized by G protein-coupled receptor kinases (GRKs). However, the role of GRKs in corticotropic cells has not been determined. In this study we focused on involvement of GRKs in desensitization of CRFR1 by CRF in corticotropic cells. We found that GRK2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). To determine the role of GRK2 in CRF-induced desensitization of CRFR1 in mouse corticotrophs, AtT-20 cells were transfected with a dominant-negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRFR1. Desensitization of CRFR1 by CRF was significantly less in AtT-20 cells transfected with the dominant-negative mutant GRK2 construct compared with desensitization in control (an empty vector-transfected) AtT-20 cells. Furthermore, pretreatment with a protein kinase A inhibitor also partially blocked desensitization of CRFR1 by CRF. These results suggest that GRK2 is involved in CRF-induced desensitization of CRFR1 in AtT-20 cells, and the protein kinase A pathway may also have an important role in desensitization of CRFR1 by CRF seen in corticotropic cells.

Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 microg/mouse, i.p.) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.

Effect of Pioglitazone on Adrenocorticotropic Hormone and Cortisol Secretion in Cushing’s Disease

The lack of an effective medical therapy for patients with Cushing's disease (CD) requires the search for new modalities of treatment. Peroxisomal proliferator-activated receptors (PPAR)-gamma are abundantly expressed in ACTH-secreting pituitary tumors. Treatment with PPARgamma agonists inhibits ACTH-secreting pituitary tumor growth, proliferation, and ACTH secretion in vitro in human and murine models and in vivo in murine corticotroph tumors. It was hypothesized that treatment with the PPARgamma agonist pioglitazone would normalize the hypothalamic-pituitary-adrenal axis of patients with CD. We evaluated the hypothalamic pituitary adrenal axis in five patients with CD in whom we measured: 1) the 24-h urine concentration of free cortisol; 2) the 24-h profile of serum cortisol and plasma ACTH; and 3) the ACTH and cortisol response to CRH stimulation. All measurements were taken at baseline and after low-dose dexamethasone treatment (0.5 mg dexamethasone every 6 h). The entire protocol was done before and after 30 d of treatment with 45 mg of daily oral pioglitazone. At baseline, before low-dose dexamethasone, all five patients had elevated 24-h urine free cortisol, elevated 24-h serum cortisol and plasma ACTH levels, and robust responses to CRH, consistent with their diagnosis of CD. There was no significant change in any of the above variables after 30 d of treatment with pioglitazone. Furthermore, there was no significant difference in the number of cortisol or ACTH spikes or in their diurnal rhythms. In summary, pioglitazone treatment (45 mg daily for 30 d) of patients with CD was not found to be effective at attenuating either ACTH or cortisol levels and does not appear to be an alternative to surgical therapy.

Comparative Study of gp130 Cytokine Effects on Corticotroph AtT-20 Cells – Redundancy or Specificity of Neuroimmunoendocrine Modulators?

Objective: This comparative in vitro study examined the effects of all known gp130 cytokines on murine corticotroph AtT-20 cell function. Methods: Cytokines were tested at equimolar concentrations from 0.078 to 10 nM. Tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)3 and STAT1, the STAT-dependent suppressor of cytokine signaling (SOCS)-3 promoter activity, SOCS-3 gene expression, STAT-dependent POMC promoter activity and adrenocorticotropic hormone (ACTH) secretion were determined. Results: Leukemia inhibitory factor (LIF), human oncostatin M (OSM) and cardiotrophin (CT)-1 (LIFR/gp130 ligands), as well as ciliary neurotrophic factor (CNTF) and novel neurotrophin-1/B-cell stimulating factor-3 (CNTFRα/LIFR/gp130 ligands) are potent stimuli of corticotroph cells in vitro. In comparison, interleukin (IL)-6 (IL-6R/gp130 ligand) and IL-11 (IL-11R/gp130 ligand) exhibited only modest direct effects on corticotrophs, while murine OSM (OSMR/gp130 ligand) showed no effect. Conclusion: (i) CNTFR complex ligands are potent stimuli of corticotroph function, comparable to LIFR complex ligands; (ii) IL-6 and IL-11 are relatively weak direct stimuli of corticotroph function; (iii) differential effects of human and murine OSM suggest that LIFR/gp130 (OSMR type I) but not OSMR/gp130 (OSMR type II) are involved in corticotroph signaling. (iv) CT-1 has the hitherto unknown ability to stimulate corticotroph function, and (v) despite redundant immuno-neuroendocrine effects of different gp130 cytokines, corticotroph cells are preferably activated through the LIFR and CNTFR complexes.

Evidence from studies on co-cultures of TtT/GF and AtT20 cells that Annexin 1 acts as a paracrine or juxtacrine mediator of the early inhibitory effects of glucocorticoids on ACTH release.

Annexin 1 (ANXA1) is a key mediator of the inhibitory effects of glucocorticoids on adrenocorticotropic hormone (ACTH) release, which develop within 1-2 h of a steroid challenge. Our previous studies, which showed that (i) ANXA1 is expressed principally by the nonsecretory folliculo-stellate cells in the pituitary gland; (ii) glucocorticoids cause the exportation of ANXA1 from these cells; and (iii) corticotrophs express specific ANXA1 binding sites, led us to propose that ANXA1 serves as a paracrine or juxtacrine mediator of glucocorticoids. To address this hypothesis, we examined ANXA1-dependent glucocorticoid actions in co-cultures of murine corticotroph (AtT20 clone D1) and folliculo-stellate (TtT/GF) cell lines. ANXA1 mRNA and protein were found in abundance in TtT/GF cells but neither was detectable in the AtT20 cells. AtT20 cells (alone and in co-culture with TtT/GF cells) responded to corticotropin-releasing hormone (CRH) (0.1-1 micro m) with increased ACTH release. The CRH-stimulated release of ACTH from AtT20 cells cultured alone was unaffected by preincubation with dexamethasone (Dex, 100 nm); by contrast, in co-cultures of AtT20 and TtT/GF cells, the steroid readily inhibited the secretory response to CRH. The effects of Dex on ACTH release were mimicked by N-terminal ANXA1 fragments (ANXA1Ac2-26, 2 micro g/ml and ANXA11-188, 0.1 ng/ml) and reversed by mifepristone (1 micro m) and by an antisense oligodeoxynucleotide (ODN) to ANXA1 (50 nm) but not by control ODNs. The antisense ODN also specifically blocked the Dex-induced externalization of ANXA1 from TtT/GF cells. Immunofluorescence imaging of the co-cultures localized the exported protein to the vicinity of the AtT20 cells and identified ANXA1 binding sites on these cells. These results provide functional and histological evidence to support our premise that the early inhibitory effects of glucocorticoids on ACTH release are dependent upon paracrine/juxtacrine actions of ANXA1 derived from folliculo-stellate cells.

Novel neurotrophin-1/B cell-stimulating factor-3 (cardiotrophin-like cytokine) stimulates corticotroph function via a signal transducer and activator of transcription-dependent mechanism negatively regulated by suppressor of cytokine signaling-3.

Novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3) is a recently cloned gp130 cytokine, acting through the tripartite ciliary neurotrophic factor receptor (CNTFR) alpha/leukemia inhibitory factor receptor (LIFR)/gp130 receptor complex. The aim of the current study was to investigate the role of NNT-1/BSF-3 in corticotroph cell function and further characterize NNT-1/BSF-3 signaling pathways. Using RT-PCR, expression of ciliary neurotrophic factor receptor alpha, leukemia inhibitory factor receptor, and gp130 could be demonstrated in mRNA derived from murine corticotroph AtT-20 cells and murine pituitary tissue. Incubation of AtT-20 cells with 10 ng/ml recombinant human NNT-1/BSF-3 rapidly induced tyrosine-phosphorylation of signal transducer and activator of transcription (STAT)3 and STAT1 at 5 and 10 min. Proopiomelanocortin promoter activity and suppressor of cytokine signaling (SOCS)-3 promoter activity were significantly stimulated by NNT-1/BSF-3 4.0 +/- 0.3- and 5.9 +/- 0.2-fold, respectively. In comparison with untreated control, NNT-1/BSF-3 significantly stimulated ACTH secretion at 24 and 48 h 1.7 +/- 0.2-fold and 1.5 +/- 0.1-fold above baseline. In comparison with mock-transfected cells, stable overexpression of SOCS-3 in AtT-20 cells abolished NNT-1/BSF-3-induced STAT1 and STAT3 phosphorylation and almost completely inhibited STAT-dependent proopiomelanocortin promoter and SOCS-3 promoter activities. In addition, NNT-1/BSF-3-induced ACTH secretion at 48 h was significantly attenuated by SOCS-3 overexpression. In summary, we have shown that NNT-1/BSF-3 is a modulator of corticotroph cell function, which is negatively regulated by SOCS-3. Our data indicate that the activation of the Jak-STAT cascade is essential for corticotroph NNT-1/BSF-3 signaling. Further studies will have to investigate the possible in vivo role of NNT-1/BSF-3 as a neuroimmunoendocrine modulator of hypothalamus-pituitary-adrenal axis stress response.

Expression of various clock genes in the murine corticotroph AtT-20 cell line

Expression of various clock genes in the murine corticotroph AtT-20 cell line

Novel pituitary ligands: peroxisome proliferator activating receptor-gamma.

Pituitary tumors cause considerable morbidity due to local invasion, hypopituitarism, or hormone hypersecretion. In many cases, no suitable drug therapies are available, and surgical excision is currently the only effective treatment. We have recently demonstrated abundant expression of nuclear hormone receptor PPAR-gamma in human pituitary tumors of different subtypes. PPAR-gamma activators (thiazolidinediones) induced G0-G1 cell-cycle arrest and apoptosis in human, and murine corticotroph, somatolactotroph, and gonadotroph pituitary tumor cells, and suppressed in vitro hormone secretion. In vivo development and growth of murine corticotroph, somatolactotroph and gonadotroph tumors, generated by subcutaneous injection of ACTH-secreting AtT20, PRL- and GH-secreting GH3, and LH-secreting LbetaT2, and alpha-T3 cells, was markedly suppressed in rosiglitazone treated mice, and plasma ACTH, and serum corticosterone, GH, PRL and LH levels were attenuated in all treated animals. PPAR-gamma is an important novel molecular target in pituitary adenoma cells and as PPAR-gamma ligands inhibit tumor cell growth and ACTH, GH, PRL and LH secretion in vitro and in vivo, thiazolidinediones are proposed as a novel oral medical management for pituitary tumors.