Somatotrope Differentiation Research Articles

FOXO1 regulates expression of Neurod4 in the pituitary gland

Pituitary gland function is regulated by the activity of various transcription factors that control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for normal somatotrope differentiation and function. Recent in vivo data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. In the current study, the somatotrope-like cell line GH3 was treated with a FOXO1 inhibitor, resulting in significant reduction in Neurod4 and Gh1 expression. Consistent with these findings, CRISPR/Cas9-mediated deletion of Foxo1 in GH3 cells significantly reduced expression of Gh1 and Neurod4. Chromatin immunoprecipitation sequencing identifies novel FOXO1 binding sites associated with the Neurod4, Gh1, and Pou1f1 genes. The FOXO1 binding site in the Neurod4 gene exhibits enhancer activity in somatotrope-like cells but not in gonadotrope-like cells. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation.

FOXO Transcription Factors Are Required for Normal Somatotrope Function and Growth.

Understanding the molecular mechanisms underlying pituitary organogenesis and function is essential for improving therapeutics and molecular diagnoses for hypopituitarism. We previously found that deletion of the forkhead factor, Foxo1, in the pituitary gland early in development delays somatotrope differentiation. While these mice grow normally, they have reduced growth hormone expression and free serum insulin-like growth factor-1 (IGF1) levels, suggesting a defect in somatotrope function. FOXO factors show functional redundancy in other tissues, so we deleted both Foxo1 and its closely related family member, Foxo3, from the primordial pituitary. We find that this results in a significant reduction in growth. Consistent with this, male and female mice in which both genes have been deleted in the pituitary gland (dKO) exhibit reduced pituitary growth hormone expression and serum IGF1 levels. Expression of the somatotrope differentiation factor, Neurod4, is reduced in these mice. This suggests a mechanism underlying proper somatotrope function is the regulation of Neurod4 expression by FOXO factors. Additionally, dKO mice have reduced Lhb expression and females also have reduced Fshb and Prl expression. These studies reveal FOXO transcription factors as important regulators of pituitary gland function.

Role of FOXO1 in Glucocorticoid-Induced Somatotrope Maturation

Growth hormone (GH) is a well-known metabolic factor secreted by pituitary somatotropes. Transcription factors such as POU1F1 and NEUROD4 promote somatotrope differentiation, maturation, and function. The forkhead transcription factor, FOXO1, is necessary for the proper timing of somatotrope differentiation and function, but the underlying mechanisms behind it have yet to be unraveled. Pituitary gland development also depends on regulation by signaling factors and hormones. Glucocorticoids have mixed effects on growth hormone production. However, when the effects of glucocorticoid signaling on the hypothalamus and pituitary gland are uncoupled, the direct effects of glucocorticoid signaling on pituitary somatotropes are not only stimulatory, but necessary for initiation of somatotrope maturation and for maintenance of somatotrope function. We find that FOXO1 is necessary for glucocorticoid induction of important somatotrope genes. Activation of glucocorticoid signaling in the somatotrope-derived MtT/S cell line induces transient expression of the bZIP transcription factor, Crebl2 within 2 hours. Interestingly, glucocorticoid induction of Crebl2 as well as the somatotrope genes Ghrhr and Gh1, is impaired in the presence of the FOXO1 inhibitor (AS1842856). There are several possible mechanisms underlying the requirement of FOXO1 in glucocorticoid induction of somatotrope maturation. One possible mechanism is that glucocorticoid signaling upregulates expression of Foxo1 and ultimately FOXO1 targets. Consistent with this possibility, Foxo1 expression is induced 8 hours after activation of glucocorticoid signaling. This does not appear to be the only mechanism underlying the role for FOXO1 in mediating glucocorticoid-induced somatotrope maturation, however, because many FOXO1 target genes, such as Neurod4 and Fosl2 are not affected by glucocorticoid signaling. We are currently investigating whether cooperative binding between FOXO1 and the glucocorticoid receptor contributes to transcriptional regulation of common targets genes. Together these data demonstrate that FOXO1 is a key factor mediating glucocorticoid induction of somatotrope maturation.

MON-483 FOXO1 Contributes to Somatotrope Differentiation through Regulation of Key Transcription Factors

Pituitary gland function is regulated by the activity of various transcription factors which control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for the proper timing of somatotrope differentiation and for somatotrope function, but the exact mechanism of action has yet to be elucidated. Recent data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. Previously, a mouse model with conditional deletion of Foxo1 from the developing pituitary gland displayed reduced Gh1 and Neurod4 transcripts as early as embryonic day 18.5. Additional data from adult animals with conditional deletion of both Foxo1 and Foxo3 from the pituitary gland have a similar reduction in Neurod4 and Gh1, as well as Pou1f1. In order to investigate the mechanism by which FOXO1 regulates pituitary gland gene expression and confirm in vivo findings, the somatotrope-like cell line MtT/S was treated with FOXO1 inhibitor, AS184256, for 24 hours at various concentrations. Neurod4 and Pou1f1 were the most severely affected genes with a dose-dependent reduction in transcript at inhibitor concentrations as low as 30 nM (p < 0.05). Gh1 transcripts were significantly reduced at 3 μM (p < 0.01). To ascertain whether FOXO1 binds to regulatory regions of key somatotrope genes, ChIP-seq was performed using the MtT/S cell line and anti-FOXO1 antibody. This study identified FOXO1 interaction with genomic regions associated with Neurod4, Pou1f1, and Gh1. Interestingly, the FOXO1 binding intervals associated with Neurod4 and Pou1f1 are highly conserved between rat and human, indicating these sequences are important for survival. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation. Investigation is also underway to identify proteins within the FOXO1 transcriptional complex. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was performed using the MtT/S cell line for this purpose. Proteins important for signal transduction and transcriptional regulation, including PKC, LRPPRC, and YBX1, were enriched in chromatin containing FOXO1, suggesting that they contribute to transcriptional control of FOXO1 targets. Overall, the emerging mechanism for FOXO1 control of somatotrope differentiation likely involves direct regulation of Neurod4, Pou1f1, and Gh1. These novel findings contribute to the much-needed understanding of pituitary cell fate decisions.

Premature Expression of FOXO1 in Developing Mouse Pituitary Results in Anterior Lobe Hypoplasia.

The process by which the somatotrope lineage emerges in the developing pituitary is regulated by the activity of specific signaling and transcription factors expressed during development. We set out to understand the contribution of FOXO1 to that process by using a mouse model in which FOXO1 is prematurely expressed in the pituitary primordium. Expression of FOXO1 in the oral ectoderm as early as embryonic day (e)9.5 resulted in pituitary gland hypoplasia and reduced expression of anterior lobe hormone transcripts at e18.5. Of note, the relative numbers of somatotropes and thyrotropes were also decreased at e18.5. LHX3 and PITX2, markers of pituitary identity, were present in a reduced number of cells during the formation of the Rathke pouch. Thus, premature expression of FOXO1 may affect adoption of pituitary identity during differentiation. Our results demonstrate that the timing of FOXO1 activation affects its role in pituitary gland organogenesis and somatotrope differentiation.

Foxo1 Is Required for Normal Somatotrope Differentiation.

The etiology for half of congenital hypopituitarism cases is unknown. Our long-term goal is to expand the molecular diagnoses for congenital hypopituitarism by identifying genes that contribute to this condition. We have previously shown that the forkhead box transcription factor, FOXO1, is present in approximately half of somatotropes at embryonic day (e) 18.5, suggesting it may have a role in somatotrope differentiation or function. To elucidate the role of FOXO1 in somatotrope differentiation and function, Foxo1 was conditionally deleted from the anterior pituitary (Foxo1Δpit). Uncommitted progenitor cells are maintained and able to commit to the somatotrope lineage normally based on the expression patterns of Sox2, a marker of uncommitted pituitary progenitors, and Pou1f1 (also known as Pit1), which marks committed progenitors. Interestingly, Foxo1Δpit embryonic mice exhibit delayed somatotrope differentiation as evidenced by an almost complete absence of GH immunoreactivity at e16.5 and reduced expression of Gh at e18.5 and postnatal day (P) 3. Consistent with this conclusion, expression of GHRH receptor, a marker of terminally differentiated somatotropes, is significantly reduced at e18.5 and P3 in the absence of FOXO1. The mechanism of FOXO1 regulation of somatotrope differentiation may involve the basic helix-loop-helix transcription factor, Neurod4, which has been implicated in somatotrope differentiation and is significantly reduced in Foxo1Δpit mice. Foxo1Δpit mice do not exhibit growth defects, and at P21 their pituitary glands exhibit a normal distribution of somatotropes. These studies demonstrate that FOXO1 is important for initial somatotrope specification embryonically but is dispensable for postnatal somatotrope expansion and growth.

Pituitary Regeneration: It'll Knock Your SOX Off!

Tissue regeneration provides promise for improved treatments for many human pathologies affecting neural and cardiac tissues, pancreas, bone, and cochlear hair cells (1–5). Recent studies in the pituitary gland have demonstrated that although cell turnover is normally low, pituitary stem cells do exist and can regenerate pituitary endocrine cell types in response to physiological stressors (6, 7). Increasing our understanding of and ability to manipulate pituitary regeneration is important for improving therapeutics for patients with hypopituitarism. Pituitary stem cells reside primarily in the marginalzone, a layer of cells bordering the pituitary cleft, and express several stem cell markers, including sex-determining region Y box 2 (SOX2) (6, 8, 9). Additional stem cell niches may exist in the pituitary gland as a subset of SOX2 cells are scattered throughout the anterior pituitary (6, 7, 10, 11). Deletion of Sox2 causes severe pituitary hypoplasia and reduced differentiation of somatotropes and thyrotropes due to insufficient renewal of periluminal stem cells (12). Additional evidence for the presence of pituitary stem cells comes from studies showing that SOX2 pituitary cells from embryos and adults have the ability to differentiate into multiple pituitary endocrine cell types in culture and in vivo, suggesting that these cells are multipotent (8, 13). In this issue of Endocrinology, an elegant study by Willems et al (33) provides insight into pituitary regeneration. Previously, this group used a transgenic mouse model (GHCre/iDTR) to target somatotropes for ablation by diphtheria toxin (DT) (14). Treatment with DT for 3 days obliterated nearly all somatotropes. In young adult mice, somatotropes were able to regenerate, and somatotrope populations were partially restored. The process of regeneration involved expansion of the SOX2 marginal-zone niche and the appearance of cells doubly positive for cytoplasmic SOX2 and GH, which likely represent cells transitioning from progenitors to terminally differentiated somatotropes (14). In the current study, Willems et al (33) expand their investigation by addressing the role of recovery period and age in regenerative capacity. Using the same model (GHCre/iDTR), they demonstrate that increasing the recovery period to 19 months does not significantly increase restoration of somatotrope numbers. Interestingly, after ablation of somatotropes in older mice, regeneration no longer occurrs, suggesting that regenerative capacity of the pituitary gland is lost with age. The loss of regenerative capacity in older animals correlates with a significant reduction in stem cells, especially those containing nuclear SOX2. Thus, nuclear SOX2 may be an indicator of healthy stem cells that are able to contribute to regenerative capacity. To determine how pituitary stem cells respond to extended ablation of somatotropes, Willems et al (33) treated young GHCre/iDTR mice with DT for 10 days. Interestingly, they find that somatotrope regeneration does not occur under these conditions, suggesting that continual ablation of somatotropes exhausts regenerative capacity of stem cells in young adult pituitary glands. Stem cell exhaustion has been observed in other systems as well. Deletion of the forkhead transcription factor family members, Foxo1, Foxo3, and Foxo4, in neural stem cells of mice results in aberrant proliferation and increased brain size. The excessive proliferation of neural stem cells eventually leads to exhaustion of their regenerative capacity ultimately causing a reduction in neural stem cells and neural degeneration (15). These data suggest that regulation of proliferation is important for maintaining stem cell health and regenerative capacity. Similar results were observed with deletion of Foxo family members in hemato-

The dynamic chromatin configuration during pituitary differentiation (562.1)

Pit‐1, a pituitary specific POU homeodomain transcription factor, plays an essential role in the process of differentiation of anterior pituitary. Abolishing the expression of Pit‐1 results in the combined block in the differentiation of somatotropes, lactotropes, and thyrotropes in anterior pituitary. Prior studies have revealed that the initial activation of Pit‐1 at embryonic day 14 (E14) in the developing mouse pituitary. This activation is mediated by the binding of transcription factor Atbf1 at the ‘early enhancer’ of Pit‐1, located ‐5.8 kb upstream of the Pit‐1 promoter, and by concurrent binding of Prop‐1 at the promoter. The maintenance phase of Pit‐1 expression is initiated at E16.5 by an auto‐regulatory mechanism in which Pit‐1 binds to its own promoter and to a distal enhancer (‘late enhancer’) located 10 kb upstream of the Pit‐1 promoter. Despite the identification of these regulatory elements, the accompanying changes in chromatin architecture at the Pit‐1 locus during the pituitary development remains undefined. In the current study, we investigate the chromatin configuration of the Pit‐1 locus at different developmental stages. We detect acetylation that is restricted to the early enhancer and the promoter at E14.5 while acetylation spreads throughout the entire locus at the adult stage. Our 3C assays reveal that the early enhancer is positioned proximal to the Pit‐1 promoter while in the maintenance phase is accompanied by a switch in the locus conformation, with positioning of the distal enhancer in proximity to the promoter. Furthermore, our studies suggest that the interaction between the distal enhancer and the promoter is mediated by Pit‐1 and CBP in differentiated pituitary. Taken together, our studies demonstrate that the regulation of Pit‐1 expression is accompanied by the dynamic alternation of the chromatin conformation during the pituitary differentiation and the chromatin architectures play important roles in activation and maintenance of gene expression.Grant Funding Source: Supported by University Research foundation of University of Pennsylvania

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Hypothalamic and hypophyseal regulation of growth hormone secretion.

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.

Growth hormone secretion: The role of glucocorticoids

Glucocorticoids (GCs) modulate the somatotropic axis at a genomic and a non-genomic level. Critical concentrations of steroids not only determine somatotrope differentiation but also enhance growth hormone (GH) gene expression. At a cellular level GH-releasing hormone (GHRH) and somatostatin (SS) are the two principal neuropeptides involved in the release of GH. In vitro data indicates that steroids enhance GH release by altering the affinity and the density of GHRH receptors. In addition, they reduce the sensitivity of the somatotrope to SS and decrease IGF-1 induced negative feedback on GH secretion. The net effect is an enhancement of GH release.

Differentiation of fetal rat somatotropes in vitro: effects of cortisol, 3,5,3'-triiodothyronine, and glucagon, a light microscopic and radioimmunological study.

Cortisol stimulates somatotrope differentiation in vitro. T3 and/or glucagon may also be involved. Fetal rat pituitary primordia were explanted at 14 days gestation and cultured for 7 days in medium supplemented with cortisol (50-500 nM), and either T3 (0.67 nM) or glucagon (0.5 nM). Also, to determine the time of first appearance of the somatotropes, explants were cultured 4, 5, or 6 days with cortisol alone. Immunoreactive somatotropes were detected by immunohistochemistry, and their size and number were estimated for each medium. GH was measured by RIA in explants and media. Immunoreactive somatotropes first appear at 18-19 days gestation. Their size and number depend on cortisol concentration: no cells at 50 nM, a few small ones at 100 nM, and many large ones at 250-500 nM. This progression was reflected by RIA of GH in explants and media, although small quantities were detected with 50 nM. The effect of T3 was only visible with a low dose of cortisol. With 100 nM cortisol, it increased the size and number of cells. Differentiation was also triggered with 50 nM cortisol plus T3. RIA detected significantly higher GH content and secretion after T3 stimulation. The decreases in number, size, and GH secretion and content elicited by glucagon were not significant, probably due to the high variability. Both techniques used provide similar information on somatotrope differentiation: stimulation by cortisol alone, or alternatively by a synergistic action between cortisol and T3.

Fetal rat somatotropes in vitro: effects of insulin, cortisol, and growth hormone-releasing factor on their differentiation: a light and electron microscopic study.

Somatotropes first appear in the fetal rat pituitary just before term. These cells have never been detected in cultured fetal pituitaries. A modified culture medium has, however, enabled their differentiation in vitro. Hypophysial primordia were explanted on days 13-18 of gestation and cultured in different media until the equivalent of term. Immunoreactive somatotropes could be detected, by light and electron microscopy, in cultured primordia explanted on day 14 of gestation or later. The size and numbers of immunoreactive cells depended on culture medium composition. The control medium, containing insulin, cortisol, T3, and glucagon, proved favorable to somatotrope differentiation and proliferation. Increased insulin concentration reduced somatotrope numbers. In the presence of only insulin and cortisol (or corticosterone) somatotropes were more numerous than in the control. Culture medium enriched with insulin alone, with insulin and T3, or with insulin and glucagon, was not suitable for development of this cell type. Addition of GH-releasing factor ( GHRF ) to the medium during the first culture day did not accelerate the first appearance of the somatotropes but did significantly increase their size. GHRF addition 1/2 h before the end of culture did not modify their morphology. The ultrastructure of somatotropes in vitro is very similar to that observed in vivo on day 21 of gestation. The cells were characterized by their lamellar endoplasmic reticulum and immunoreactive secretory granules (300-400 nm maximal section diameter). Fetal somatotropes can, therefore, be successfully caused to differentiate in vitro. Their appearance depends on insulin and glucocorticoid concentration. T3 and/or glucagon may be inhibitory. GHRF may increase storage in somatotropes. These factors may also regulate the development of somatotropes in vivo.