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 Fkbp4 and Fkbp5 on Regulation of the Proopiomelanocortin Gene in Murine AtT-20 Corticotroph Cells.

The hypothalamic-pituitary-adrenal axis is stimulated in response to stress. When activated, it is suppressed by the negative feedback effect of glucocorticoids. Glucocorticoids directly inhibit proopiomelanocortin (Pomc) gene expression in the pituitary. Glucocorticoid signaling is mediated via glucocorticoid receptors, 11ß-hydroxysteroid dehydrogenases, and the FK506-binding immunophilins, Fkbp4 and Fkbp5. Fkbp4 and Fkbp5 differentially regulate dynein interaction and nuclear translocation of the glucocorticoid receptor, resulting in modulation of the glucocorticoid action. Here, we explored the regulation of Fkbp4 and Fkbp5 genes and their proteins with dexamethasone, a major synthetic glucocorticoid drug, in murine AtT-20 corticotroph cells. To elucidate further roles of Fkbp4 and Fkbp5, we examined their effects on Pomc mRNA levels in corticotroph cells. Dexamethasone decreased Pomc mRNA levels as well as Fkpb4 mRNA levels in mouse corticotroph cells. Dexamethasone tended to decrease Fkbp4 protein levels, while it increased Fkpb5 mRNA and its protein levels. The dexamethasone-induced decreases in Pomc mRNA levels were partially canceled by Fkbp4 knockdown. Alternatively, Pomc mRNA levels were further decreased by Fkbp5 knockdown. Thus, Fkbp4 contributes to the negative feedback of glucocorticoids, and Fkbp5 reduces the efficiency of the glucocorticoid effect on Pomc gene expression in pituitary corticotroph cells.

Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans.

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.

Hypothalamic Contribution to Pituitary Functions Is Recapitulated In Vitro Using 3D-Cultured Human iPS Cells.

The pituitary is a major hormone center that secretes systemic hormones responding to hypothalamus-derived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs). Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a life-threatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.

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.

The retrotransposon gag domain containing protein Rgag4 is an Ikaros target in the pituitary.

Previous studies have established the common and critical involvement of the zinc finger protein Ikaros in lymphoid and pituitary cell development and expansion. Key to the assembly of several transcriptional networks, we have demonstrated up-regulation of Ikaros and its interacting partner the C-terminal Binding Protein (CtBP) in response to hypoxia. This prompted us to explore common transcriptional targets using a chromatin immunoprecipitate (ChIP) screen of DNA from pituitary corticotroph cells. This strategy yielded a finite list of targets common to both transcription factors that included the metalloprotease ADAMTS10. In this report, we focus on validation of a second candidate target, the retrotransposon gag domain containing protein Rgag4. We identified the ability of Ikaros to bind the Rgag4 promoter, influence its transcriptional activity, and induce endogenous gene expression. Robust expression of Rgag4 was noted in the anterior lobe of the pituitary gland which was diminished in Ikaros knockout mice. Down-regulation of Rgag4 resulted in profound reduction of hormone gene expression with diminished ACTH secretion, recapitulating the effect of Ikaros deficiency in knockout mice. The results introduce Rgag4 to the repertoire of effectors serving to couple the chromatin remodeler Ikaros with the hormonal stress response.

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion.

Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.

ATAT1 is essential for regulation of homeostasis-retaining cellular responses in corticotrophs along hypothalamic-pituitary-adrenal axis.

The production and secretion of adrenocorticotropin, a proopiomelanocortin (POMC)-derived hormone, by corticotrophs in the anterior pituitary, is regulated by corticotrophin-releasing hormone (CRH) and glucocorticoids. We have previously demonstrated that adrenalectomy induces α-tubulin N-acetyltransferase 1 (ATAT1) expression and α-tubulin acetylation in corticotrophs. However, the regulatory mechanism of ATAT1 expression and the function of acetylated microtubules in corticotrophs are unclear. Here, we analyze the effect of CRH or dexamethasone on Atat1 expression in the mouse corticotroph AtT20 cell line. The expression of Atat1 was increased by CRH and decreased by dexamethasone in AtT20 cells. We examined the effect of Atat1 knockdown on the expression of POMC-associated genes and the dexamethasone-induced nuclear translocation of glucocorticoid receptor (GR) by real-time polymerase chain reaction and Western blot analysis, respectively. Atat1 knockdown resulted in a significant increase in the expression of ACTH-producing genes and decreased the dexamethasone-induced nuclear translocation of GR accompanied with a reduction in α-tubulin acetylation. Atat1 overexpression resulted in a significant increase in α-tubulin acetylation and the dexamethasone-induced nuclear translocation of GR. These results suggest that the acetylated microtubules function as the rail-line for the transportation of GR into the nucleus. We conclude that ATAT1 finely tunes the cellular responses of corticotrophs to hormonal stimulation through an intracellular feedback circuit.

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.

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.

Adrenalectomy facilitates ATAT1 expression and α-tubulin acetylation in ACTH-producing corticotrophs.

Microtubules play an important role in the intracellular transport of secretory granules in endocrine cells and in mitosis and the maintenance of cell morphology and are composed of heterodimers of α- and ß-tubulin. α-Tubulin N-acetyltransferase 1 (ATAT1), which acetylates the lysine residue at position 40 of α-tubulin, functions not only in stabilizing microtubule structures and forming the primary cilium assembly but also in vesicular trafficking in neurons. However, the localization of ATAT1 and the role of α-tubulin acetylation in endocrine cells in the pituitary are still poorly understood. Corticotrophs in the anterior lobe of the pituitary produce and secrete adrenocorticotropin (ACTH). Although removal of the adrenal gland, a target organ of ACTH, is reported to promote the synthesis and secretion of ACTH in corticotrophs and to induce structural alterations in their organelles, uncertainty remains as to whether the acetylation of α-tubulin is involved in such intracellular events of corticotrophs. We investigate the expression and localization of ATAT1 and the acetylation of α-tubulin in the pituitary of normal and adrenalectomized rats. We find that ATAT1 is localized to the Golgi apparatus of endocrine cells in the anterior lobe of normal pituitary and that the expression levels of ATAT1 and acetylation levels of α-tubulin increase following adrenalectomy. These results agree with the hypothesis that the acetylation of α-tubulin by ATAT1 regulates the intracellular transport of secretory granules in corticotrophs.

SOX2 is sequentially required for progenitor proliferation and lineage specification in the developing pituitary.

Sox2 mutations are associated with pituitary hormone deficiencies and the protein is required for pituitary progenitor proliferation, but its function has not been well characterized in this context. SOX2 is known to activate expression of Six6, encoding a homeodomain transcription factor, in the ventral diencephalon. Here, we find that the same relationship likely exists in the pituitary. Moreover, because Six6 deletion is associated with a similar phenotype as described here for loss of Sox2, Six6 appears to be an essential downstream target of SOX2 in the gland. We also uncover a second role for SOX2. Whereas cell differentiation is reduced in Sox2 mutants, some endocrine cells are generated, such as POMC-positive cells in the intermediate lobe. However, loss of SOX2 here results in complete downregulation of the melanotroph pioneer factor PAX7, and subsequently a switch of identity from melanotrophs to ectopic corticotrophs. Rescuing proliferation by ablating the cell cycle negative regulator p27 (also known as Cdkn1b) in Sox2 mutants does not restore melanotroph emergence. Therefore, SOX2 has two independent roles during pituitary morphogenesis; firstly, promotion of progenitor proliferation, and subsequently, acquisition of melanotroph identity.

Dmrt5 controls corticotrope and gonadotrope differentiation in the zebrafish pituitary.

Dmrt transcription factors control sex determination or sex-specific differentiation across all invertebrate and vertebrate species, in which they have been studied so far. In addition to important functions in the reproductive system, also nongonadal roles have been assigned to several dmrt family members. One example is dmrt5, which was shown to guide neurogenesis in the forebrain of some vertebrates including fish. Here we show that in zebrafish, dmrt5 is also expressed adjacent to the pituitary anlage and later in the anterior pars distalis in which it organizes differentiation of endocrine cells. We find that pituitary induction, cell survival, proliferation, and early lineage specification in the pituitary is independent of dmrt5. Instead, dmrt5 is required for terminal differentiation of corticotropes and gonadotropes. Gene knockdown and mutant analysis revealed that dmrt5 promotes corticotrope differentiation via tbx19 expression, whereas it prevents gonadotrope differentiation in the anterior pars distalis. In dmrt5 morphants and mutants, reduced corticotrope numbers may result in irregular positioning and reduced maintenance of lactotropes. In conclusion, our study establishes a novel function for dmrt5 for cell differentiation in the anterior pituitary. Intriguingly, its effect on gonadotrope numbers defines a first nongonadal role for a dmrt family member that appears crucial for the activity of the reproductive system.

Activation of the dopamine receptor type-2 (DRD2) promoter by 9-cis retinoic acid in a cellular model of Cushing's disease mediates the inhibition of cell proliferation and ACTH secretion without a complete corticotroph-to-melanotroph transdifferentiation.

Cushing's disease (CD) is a rare condition in which hypercortisolemia is secondary to excessive ACTH release from a pituitary corticotroph adenoma. CD is associated with significant morbidity and mortality, and a safe therapy that effectively targets the pituitary tumor is still lacking. Retinoic acid (RA) and dopamine agonists (DAs) have recently been considered as monotherapy in CD patients, and satisfactory results have been reported, albeit in a limited number of patients. Given the permissive role of RA on the dopamine receptor type-2 (DRD2), the aim of the present study was to see whether a combination of 9-cis RA and the DA bromocriptine (Br) might represent a possible treatment for CD. Here we show that 9-cis RA induces a functional DRD2 in the pituitary corticotroph cell line AtT20, and increases cell sensitivity to Br via a mechanism only partially related to corticotroph-to-melanotroph transdifferentiation. In addition, 9-cis RA and Br act synergistically to modulate cell viability, with favorable implications for clinical use. In nearly 45% of corticotropinoma-derived primary cultures, the combined administration of 9-cis RA and Br lowered the steady-state level of the ACTH precursor proopiomelanocortin (POMC) more efficiently than either of the drugs alone. In conclusion, the effects of a combination of 9-cis RA and Br on ACTH synthesis/secretion and cell viability in AtT20, and on POMC transcriptional activity in human corticotropinomas might represent a suitable starting point for assessing the potential of this treatment regimen for ACTH-secreting pituitary adenomas. This study thus has potentially important implications for novel therapeutic approaches to CD.

Ontogenic development of corticotrophs in fetal buffalo (Bubalus bubalis) pituitary gland.

To evaluate the subpopulation of corticotrophs in developing buffalo (Bubalus bubalis) fetus, recovered pituitary glands (n=6 per group) from late first, second and third gestational female buffalo dams. The corticotrophs were identified by using specific antibodies against proopiomelanocortin (POMC) and adrenocorticotrophic hormone (ACTH) through immunohistochemistry. There was a significant (P≤0.05) increase of immunoreactive (ir) ir-ACTH cells during late 2nd trimester while, ir-POMC cells were more (P≤0.05) at late 3rd trimester of gestation as compared to other age groups. The quantity of co-localized cells for POMC and ACTH was significantly (P≤0.05) greater at the end of 1st gestation rather than 2nd and 3rd gestational fetal adenohypophyseal cells. This study is the first to demonstrate co-localization of POMC+ACTH and the affect of gestational age on the expression of these cells in buffalo fetus adenohypophysis.

Sensorimotor cortex ablation induces time-dependent response of ACTH cells in adult rats: behavioral, immunohistomorphometric and hormonal study.

Traumatic brain injury (TBI) represents a serious event with far reaching complications, including pituitary dysfunction. Pars distalis corticotropes (ACTH cells), that represent the active module of hypothalamo-pituitary-adrenocortical axis, seem to be affected as well. Since pituitary failure after TBI has been associated with neurobehavioral impairments the aim of this study was to evaluate the effects of TBI on recovery of motor functions, morphology and secretory activity of ACTH cells in the pituitary of adult rats. Wistar male rats, initially exposed to sensorimotor cortex ablation (SCA), were sacrificed at the 2nd, 7th, 14th and 30th days post-surgery (dps). A beam walking test was used to evaluate the recovery of motor functions. Pituitary glands and blood were collected for morphological and hormonal analyses. During the first two weeks post-injury increased recovery of locomotor function was detected, reaching almost the control value at day 30. SCA induces significant increase of pituitary weights compared to their time-matched controls. The volume of ACTH-immunopositive cells was reduced at the 7th dps, while at the 14th dps their volume was enlarged, in comparison to corresponding sham controls. Volume density of ACTH cells was increased only at 14th dps, while at day 30 this increase was insignificant. The plasma level of ACTH transiently increased after the injury. The most pronounced changes were observed at the 7th and 14th dps, and were followed by decrease toward control levels at the 30th dps. Thus, temporal changes in the hypothalamic-pituitary-adrenal axis after traumatic brain injury appear to correlate with the recovery process.

Adiponectin regulates ACTH secretion and the HPAA in an AMPK-dependent manner in pituitary corticotroph cells.

It is known that adipokines can regulate the hypothalamic-pituitary-adrenal axis (HPAA). In this study, we confirmed that adiponectin regulates the HPAA by affecting pituitary corticotroph cells. Using RT-PCR and immunofluorescence, we determined that adiponectin receptors were expressed in pituitary corticotroph tumour cells (AtT-20 cells and human corticotroph tumours). Adiponectin stimulated calcium influx and increased basal ACTH secretion without affecting corticotrophin-releasing hormone (CRH)-stimulated ACTH secretion, which was most likely due to the expression of adiponectin repressing CRH receptor 1 (CRHR1). Adiponectin also acutely stimulated ACTH release in primary culture pituitary cells. Lastly, adiponectin directly phosphorylated 5' AMP-activated protein kinase (AMPK) in AtT-20 cells. The effects of adiponectin were mimicked by AICAR, which was blocked by compound C. Taken together, our results suggested that adiponectin stimulated ACTH secretion and down-regulated CRHR1, possibly via an AMPK-dependent mechanism in pituitary corticotroph cells.

Modified S/MAR episomal vectors for stably expressing fluorescent protein-tagged transgenes with small cell-to-cell fluctuations.

We modified and tested scaffold/matrix attachment region (S/MAR) episomal vectors. The new vectors would be useful in obtaining cells stably expressing fluorescent protein-tagged transgenes with small, mostly within 10-fold cell-to-cell fluctuations. In the vectors, the same transcript directs episomal replication and expression of transgene/antibiotic marker, and only antibiotic selection without any other extra steps was sufficient to obtain desired stable cells, including those expressing two different proteins simultaneously. Furthermore, the two test cases (expression of human growth hormone in AtT20 and four protein kinase C isoforms in HEK293) would prove to be useful in visualizing and analyzing regulatory processes involving these proteins.

Mitotane reduces human and mouse ACTH-secreting pituitary cell viability and function.

Medical therapy for Cushing's disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushing's syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

Adult pituitary cell maintenance: lineage-specific contribution of self-duplication.

The identification of a stable pool of progenitor/stem cells in the adult pituitary has renewed the interest of identifying mechanisms for maintenance of pituitary cells throughout life. Whereas developmental studies have shown that progenitor expansion is the major source of new differentiated cells during pituitary organogenesis, the contribution of these progenitors for maintenance of the adult tissue is not clear although progenitors were clearly involved in cell expansion following end-organ ablation, notably after adrenalectomy and/or gonadectomy. We have used a genetic trick that eliminates dividing cells by apoptosis in order to assess the contribution of differentiated corticotropes and melanotropes for maintenance of their population in the adult pituitary. The system relies on chromosome instability created by the action of the Cre recombinase on inverted loxP sites. Expression of Cre recombinase in corticotropes and melanotropes led to progressive loss of corticotropes whereas melanotropes were unaffected. Because the Cre transgene is not expressed in progenitors, the data indicate that maintenance of the adult corticotrope pool is primarily due to self-duplication of differentiated cells. In contrast, melanotropes do not divide. Maintenance of corticotropes by self-duplication contrasts with the reported proliferative response of undifferentiated cells observed after adrenalectomy. If corticotrope reentry into cell cycle constitutes a normal mechanism to maintain the adult corticotrope pool, this same mechanism may also be perturbed during corticotrope adenoma development in Cushing's disease.