Inhibition of Heat Shock Factor 1 Enhances Repressive Molecular Mechanisms on the POMC Promoter.

BACKGROUND: Cushing's disease (CD) is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. They express high levels of heat shock protein 90 and heat shock factor 1 (HSF1) in comparison to the normal tissue counterpart, indicating activated cellular stress. AIMS: Our objectives were: (1) to correlate HSF1 expression with clinical features and hormonal/radiological findings of CD, and (2) to investigate the effects of HSF1 inhibition as a target for CD treatment. PATIENTS/METHODS: We examined the expression of total and pSer326HSF1 (marker for its transcriptional activation) by Western blot on eight human CD tumours and compared to the HSF1 status of normal pituitary. We screened a cohort of 45 patients with CD for HSF1 by immunohistochemistry and correlated the HSF1 immunoreactivity score with the available clinical data. We evaluated the effects of HSF1 silencing with RNA interference and the HSF1 inhibitor KRIBB11 in AtT-20 cells and four primary cultures of human corticotroph tumours. RESULTS: We show that HSF1 protein is highly expressed and transcriptionally active in CD tumours in comparison to normal pituitary. The immunoreactivity score for HSF1 did not correlate with the typical clinical features of the disease. HSF1 inhibition reduced proopiomelanocortin (Pomc) transcription in AtT-20 cells. The HSF1 inhibitor KRIBB11 suppressed ACTH synthesis from 75% of human CD tumours in primary cell culture. This inhibitory action on Pomc transcription was mediated by increased glucocorticoid receptor and suppressed Nurr77/Nurr1 and AP-1 transcriptional activities. CONCLUSIONS: These data show that HSF1 regulates POMC transcription. Pharmacological targeting of HSF1 may be a promising treatment option for the control of excess ACTH secretion in CD.

Sirt1 inhibits the transcription factor CREB to regulate pituitary growth hormone synthesis.

Growth hormone (GH) is a major anabolic hormone and the primary regulator of organism growth. Its transcription is triggered by GH-releasing hormone (GHRH) through the transcription factor cAMP response element-binding protein (CREB) and by caloric intake. In contrast, the deacetylase Sirt1 is activated by caloric restriction. Therefore, the present study investigates how Sirt1 affects CREB function and GH synthesis. Sirt1 pharmacological activation with resveratrol (IC50=87 µM) suppressed GHRH-induced GH secretion from rat anterior pituitary cells in vivo and in vitro, while vehicle controls showed no effect. Resveratrol's effects were abolished after knocking down Sirt1 with RNA interference, but not in control scrambled siRNA-transfected rat somatotrophs, confirming the Sirt1 specificity. Sirt1 activation and overexpression suppressed forskolin-induced CREB-Ser(133) phosphorylation, but no effect was seen with vehicle and empty plasmid controls. The deacetylase-dead mutant Sirt1 retained CREB-Ser(133) phosphorylation by keeping protein phosphatase protein phosphatase 1 activity low. Sirt1 activation suppressed glycogen synthase kinase 3 ß acetylation, and a mutation on the GSK3ß-Lys(205) residue mimicking a hypoacetylated form revealed increased activity. In summary, this is a novel mechanism through which Sirt1 intercepts the cAMP pathway by suppressing CREB transcriptional activation, resulting in decreased GH synthesis.

Deficiency of FK506-binding protein (FKBP) 51 alters sleep architecture and recovery sleep responses to stress in mice.

FK506-binding protein 51 (FKBP51) is a co-chaperone of the glucocorticoid receptor, functionally linked to its activity via an ultra-short negative feedback loop. Thus, FKBP51 plays an important regulatory role in the hypothalamic-pituitary-adrenocortical (HPA) axis necessary for stress adaptation and recovery. Previous investigations illustrated that HPA functionality is influenced by polymorphisms in the gene encoding FKBP51, which are associated with both increased protein levels and depressive episodes. Because FKBP51 is a key molecule in stress responses, we hypothesized that its deletion impacts sleep. To study FKBP51-involved changes in sleep, polysomnograms of FKBP51 knockout (KO) mice and wild-type (WT) littermates were compared at baseline and in the recovery phase after 6-h sleep deprivation (SD) and 1-h restraint stress (RS). Using another set of animals, the 24-h profiles of hippocampal free corticosterone levels were also determined. The most dominant effect of FKBP51 deletion appeared as increased nocturnal wake, where the bout length was significantly extended while non-rapid eye movement sleep (NREMS) and rapid eye movement sleep were rather suppressed. After both SD and RS, FKBP51KO mice exhibited less recovery or rebound sleep than WTs, although slow-wave activity during NREMS was higher in KOs, particularly after SD. Sleep compositions of KOs were nearly opposite to sleep profiles observed in human depression. This might result from lower levels of free corticosterone in FKBP51KO mice, confirming reduced HPA reactivity. The results indicate that an FKBP51 deletion yields a pro-resilience sleep phenotype. FKBP51 could therefore be a therapeutic target for stress-induced mood and sleep disorders.

Cytokines and genes in pituitary tumorigenesis: RSUME role in cell biology.

Cytokines of the IL-6 or gp130 family regulate many cellular responses and play regulatory roles in numerous tissues, and are placed as auto-paracrine regulators of pituitary function acting in normal and tumoral anterior pituitary cells. Especially, IL-6 has a regulatory role in the hormone secretion and growth of the anterior pituitary and is involved in adenoma pathogenesis. Recently, IL-6 has been shown to mediate oncogene-induced senescence (OIS). IL-6 might participate in such a process in adenomas pituitary as well. From pituitary tumoral gp130 overexpressing cells, an unknown protein, RSUME, has been cloned. RSUME is induced by hypoxia in pituitary tumors and regulate pathways involved in angiogenic and tumorigenic processes (NF-kappaB/IkappaB and HIF-1alpha pathways). Thus, it could have an important role in the development of the pituitary tumors.

GEOFFREY HARRIS AWARD 2019: Translational research in pituitary tumours.

Although effective treatment regimens (surgical resection, drug treatment with dopamine agonists or somatostatin analogues, radiotherapy) have been established for the therapy of most pituitary tumours, a considerable proportion of affected patients cannot completely cured due to incomplete resection or drug resistance. Moreover, even if hormone levels have been normalized, patients with hormone-secreting tumours still show persistent pathophysiological alterations in metabolic, cardiovascular or neuropsychiatric parameters and have an impaired quality of life. In this review reasons for the discrepancy between biochemical cure and incomplete recovery from tumour-associated comorbidities are discussed and the clinical management is delineated exemplarily for patients with acromegaly and Cushing's disease. In view of the development of additional treatment concepts for the treatment of pituitary adenomas we speculate about the relevance of RSUME as a potential target for the development of an anti-angiogenic therapy. Moreover, the role of BMP-4 which stimulates prolactinoma development through the Smad signalling cascade is described and its role as putative drug target for the treatment of prolactinomas is discussed. Regarding the well-known resistance of a part of somatotropinomas to somatostatin analogue treatment, recently identified mechanisms responsible for the drug resistance are summarized and ways to overcome them in future treatment concepts are presented. Concerning novel therapeutic options for patients with Cushing's disease the impact of retinoic acid, which is currently tested in clinical studies, is shown, and the action and putative therapeutic impact of silibinin to resolve glucocorticoid resistance in these patients is critically discussed.

A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors.

PURPOSE: Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST2 and SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST2. However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST3-agonists and characterize their effects on experimental NFPT models. EXPERIMENTAL DESIGN: Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST3-agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST3-agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST3-agonist. RESULTS: We successfully identified the first SST3-agonist peptides. SST3-agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST3-agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST3-agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs. CONCLUSIONS: This study demonstrates that SST3-agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST3, suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Potential of retinoic acid derivatives for the treatment of corticotroph pituitary adenomas.

Cushing's disease is a severe clinical condition caused by hypersecretion of corticosteroids due to excessive ACTH secretion from a pituitary adenoma. This complex endocrine disorder still represents a major challenge for the physician in terms of efficient treatment. In the last years there was only little progress in elucidating the molecular mechanisms responsible for the constitutive and autonomous ACTH secretion of pituitary corticotrophinomas. As a consequence, no effective drug therapy is currently available, particularly if surgical excision is not successful. In the present article we examine recent studies that have investigated the therapeutic potential of retinoic acid receptors as nuclear receptor targets for the treatment of Cushing's disease. Retinoic acid is an efficient drug used for the treatment of different types of cancers and it proved to act in animal models of Cushing's disease. The efficiency of this treatment in patients with this disorder still needs to be tested in clinical trials.

Modulation of growth hormone and prolactin secretion in Trypanosoma cruzi-infected mammosomatotrophic cells.

OBJECTIVE: In a previous study, we reported an imbalance in the hypothalamus-pituitary-adrenal axis of mice acutely infected with the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. METHODS: Possible effects of this parasitic infection on the endocrine function of other pituitary cell types were studied, in particular regarding the production of prolactin (PRL) and growth hormone (GH). RESULTS: In the mammosomatotrophic cell line GH3, both GH and PRL secretion were decreased, reflecting the diminished PRL concentrations in the pituitary glands of infected mice. Additionally, expression of extracellular matrix proteins, e.g. laminin, was increased in T. cruzi-infected GH3 cells, which may be related to the diminished secretory function of these cells. Lastly, the expression of Pit-1, a major transcription factor for the PRL and GH genes, is also decreased in T. cruzi-infected cultures. CONCLUSION: T. cruzi infection downregulates PRL and GH production. Combined with our previous data showing increased glucocorticoid levels following T. cruzi infection, the immunosuppression induced by T. cruzi infection may be partially related to multiple endocrine changes involving the hypothalamus-pituitary axis and corresponding target endocrine glands.

Differential gene expression in models of pituitary prolactin-producing tumoral cells.

Although several genes and signalling pathways have been identified as important effectors in the development of pituitary tumours, our understanding of pituitary tumorigenesis remains incomplete and is the focus of much current research. Use of the mRNA differential display technique in prolactinomas from D2-receptor knockout mice and in stable GH3 cell line clones with enhanced tumorigenicity in vivo has led to the identification of two genes that are involved in the pathogenic process--BMP-4 and RSUME. Bone morphogenetic protein-4 (BMP-4) has been found to have a crucial role in prolactinoma development and also in signalling crosstalk with oestrogens. In contrast, BMP-4 has an inhibitory role in corticotrophinomas. RSUME (RWD-containing sumoylation enhancer) was identified from a transformed lactosomatotrophic cell line that had increased tumorigenic and angiogenic potential. Expression of RSUME was induced under hypoxic conditions and it has a potential role during vascularization. The differential expression and action of BMP-4 in prolactinomas and corticotrophinomas highlights the importance of studying a gene with contrasting actions in two cell lineages of the same organ in order to understand the pituitary transformation process. Both BMP-4 and RSUME may be interesting targets for inhibiting steps involved in pituitary tumorigenesis.

Regulation of pituitary function by cytokines.

Research performed on the pituitary has proven that cytokines play an important role in maintaining pituitary physiology, affecting not only cell proliferation but also hormone secretion. The effects of cytokines can be autocrine or paracrine. This review gives an overview on the effects of the most studied cytokines in the pituitary. Special interest is focused on interleukin-6 (IL-6) because it has the distinctive characteristic of stimulating pituitary tumor cell growth, but has the opposite effect on normal pituitary cells. On the other hand, IL-6 is a cytokine of interest in the pituitary because recent work has shown that it promotes and maintains senescence in certain types of tumors. Given that the majority of pituitary adenomas are microadenomas and the fact that clinically inapparent pituitary tumors are quite common, senescence, perhaps mediated by IL-6, is an attractive mechanism for explaining the benign nature of pituitary tumors.

Sleep Deprivation Alters the Pituitary Stress Transcriptome in Male and Female Mice.

Poor sleep hygiene is a growing problem, with detrimental effects on many biological systems. The pituitary gland plays a crucial role in the regulation of sleep and the stress response, and its dysfunction leads to sleep-related disorders. However, the interaction between these critical functions remains unclear. Thus, we performed a comparative, whole-transcriptome, analysis to identify stress-induced genes and relevant pathways that may be affected by sleep deprivation. One day following 12 h of Paradoxical Sleep Deprivation (PSD), mice were restrained for 20 min. Gene expression changes in the pituitary were assessed via RNA-Seq and Gene Ontology in PSD and/or restrained groups compared to controls. We show that restraint triggers transcriptional responses involved in hormone secretion, the glucocorticoid response, and apoptosis in both sexes, with 285 differentially expressed genes in females and 93 in males. When PSD preceded restraint stress, the numbers of differentially expressed genes increased to 613 in females and 580 in males. The pituitary transcriptome of restraint+PSD animals was enriched for microglia and macrophage proliferation, cellular response to corticosteroids, and apoptosis, among others. Finally, we identify sex-specific differences in restraint-induced genes following PSD. These findings provide genetic targets to consider when studying sleep and the response to stress.

Profiling of behavioral changes and hippocampal gene expression in mice chronically treated with the SSRI paroxetine.

INTRODUCTION: Monoamine-based antidepressants inhibit neurotransmitter reuptake within short time. However, it commonly takes several weeks until clinical symptoms start to resolve--indicating the involvement of effects distant from reuptake inhibition. OBJECTIVE: To unravel other mechanisms involved in drug action, a "reverse" pharmacological approach was applied to determine antidepressant-induced alterations of hippocampal gene expression. MATERIALS AND METHODS: The behavioral response to long-term paroxetine administration of male DBA/2Ola mice was assessed by the forced swim test (FST), the modified hole board (mHB), and the dark/light box. Hippocampi of test-naive mice were dissected, and changes in gene expression by paroxetine treatment were investigated by means of microarray technology. RESULTS AND DISCUSSION: Robust effects of paroxetine on passive stress-coping behavior in the FST were observed. Furthermore, anxiolytic properties of long-term antidepressant treatment could be identified in DBA mice in both, the mHB and dark/light box. Analysis of microarray results revealed a list of 60 genes differentially regulated by chronic paroxetine treatment. Preproenkephalin 1 and inhibin beta-A showed the highest level of transcriptional change. Furthermore, a number of candidates involved in neuroplasticity/neurogenesis emerged (e.g., Bdnf, Gfap, Vim, Sox11, Egr1, Stat3). Seven selected candidates were confirmed by in situ hybridization. Additional immunofluorescence colocalization studies of GFAP and vimentin showed more positive cells to be detected in long-term paroxetine-treated DBA mice. CONCLUSION: Candidate genes identified in the current study using a mouse strain validated for its responsiveness to long-term paroxetine treatment add, in our opinion, to unraveling the mechanism of action of paroxetine as a representative for SSRIs.

Octreotide, a somatostatin analogue, mediates its antiproliferative action in pituitary tumor cells by altering phosphatidylinositol 3-kinase signaling and inducing Zac1 expression.

Somatostatin limits cell growth by inhibiting the proliferative activity of growth factor receptors. In this study, it is shown that in pituitary tumor cells, the somatostatin analogue octreotide produces its antiproliferative action by inducing the expression the tumor suppressor gene Zac1. ZAC/Zac1 induces cell cycle arrest and apoptosis and is highly expressed in normal pituitary, mammary, and ovarian glands but is down-regulated in pituitary, breast, and ovarian tumors. Knocking down Zac1 by RNA interference abolished the antiproliferative effect of octreotide in pituitary tumor cells, indicating that Zac1 is necessary for the action of octreotide. The effect of octreotide on Zac1 expression was pertussis toxin sensitive and was abolished after transfection with a dominant negative vector for SHP-1. Zac1 is a target of the phosphatidylinositol 3-kinase (PI3K) survival pathway. Octreotide treatment decreased the tyrosine phosphorylation levels of the PI3K regulatory subunit p85, induced dephosphorylation of phosphoinositide-dependent kinase 1 (PDK1) and Akt, and activated glycogen synthase kinase 3beta (GSKbeta). Therefore, in pituitary tumor cells, somatostatin analogues produce their antiproliferative action by acting on the PI3K/Akt signaling pathway and increasing Zac1 gene expression.

Currently used and investigational drugs for Cushing´s disease.

INTRODUCTION: Cushing's disease (CD) is caused by a corticotroph adenoma of the pituitary gland that secretes excess adrenocorticotropic hormone (ACTH) causing increased morbidity and mortality. Surgery is the treatment of choice, but is not always successful. Alternatives include radiotherapy, adrenal surgery, and pharmaceutical therapy. The latter is increasingly gaining momentum due to the recent development of compounds that reduce hypercortisolaemia or its symptoms, acting through different mechanisms. Areas covered: In this article, the authors provide a complete overview of the treatment options for Cushing´s disease, including adrenal-directed, tumor-targeted, and peripheral therapies that are currently used or in development, and discuss their potential advantages and limitations. Expert opinion: Considering the lack of long-term remission in up to half of the patients after surgery, and the delayed response to radiotherapy along with potential side effects, there is a strong need for an effective pharmaceutical treatment. Pasireotide, mifepristone, ketoconazole and metyrapone have been approved by regulatory authorities but their use remains limited due to considerable costs and side effects. Research in this field has focused recently on the improvement of pre-existing drugs and the development of safe new ones. However, few approaches aim at targeting the source of the disease, the ACTH-secreting adenoma.

Stress-responsive FKBP51 regulates AKT2-AS160 signaling and metabolic function.

The co-chaperone FKBP5 is a stress-responsive protein-regulating stress reactivity, and its genetic variants are associated with T2D related traits and other stress-related disorders. Here we show that FKBP51 plays a role in energy and glucose homeostasis. Fkbp5 knockout (51KO) mice are protected from high-fat diet-induced weight gain, show improved glucose tolerance and increased insulin signaling in skeletal muscle. Chronic treatment with a novel FKBP51 antagonist, SAFit2, recapitulates the effects of FKBP51 deletion on both body weight regulation and glucose tolerance. Using shorter SAFit2 treatment, we show that glucose tolerance improvement precedes the reduction in body weight. Mechanistically, we identify a novel association between FKBP51 and AS160, a substrate of AKT2 that is involved in glucose uptake. FKBP51 antagonism increases the phosphorylation of AS160, increases glucose transporter 4 expression at the plasma membrane, and ultimately enhances glucose uptake in skeletal myotubes. We propose FKBP51 as a mediator between stress and T2D development, and potential target for therapeutic approaches.

Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action.

The molecular mechanisms governing the pathogenesis of ACTH-secreting pituitary adenomas are still obscure. Furthermore, the pharmacological treatment of these tumors is limited. In this study, we report that bone morphogenetic protein-4 (BMP-4) is expressed in the corticotrophs of human normal adenohypophysis and its expression is reduced in corticotrophinomas obtained from Cushing's patients compared with the normal pituitary. BMP-4 treatment of AtT-20 mouse corticotrophinoma cells has an inhibitory effect on ACTH secretion and cell proliferation. AtT-20 cells stably transfected with a dominant-negative form of the BMP-4 signal cotransducer Smad-4 or the BMP-4 inhibitor noggin have increased tumorigenicity in nude mice, showing that BMP-4 has an inhibitory role on corticotroph tumorigenesis in vivo. Because the activation of the retinoic acid receptor has an inhibitory action on Cushing's disease progression, we analyzed the putative interaction of these two pathways. Indeed, retinoic acid induces both BMP-4 transcription and expression and its antiproliferative action is blocked in Smad-4dn- and noggin-transfected Att-20 cells that do not respond to BMP-4. Therefore, retinoic acid induces BMP-4, which participates in the antiproliferative effects of retinoic acid. This new mechanism is a potential target for therapeutic approaches for Cushing's disease.

Retinoic acid as a novel medical therapy for Cushing's disease in dogs.

Cushing's disease is almost always caused by an ACTH-secreting pituitary tumor, but effective medical therapy is currently limited. Because retinoic acid has been shown to be potentially useful in decreasing corticotroph secretion and proliferation in rodent models, we have studied its action in dogs with Cushing's disease. A randomized treatment with retinoic acid (n = 22) vs. ketoconazole (n = 20) in dogs with Cushing's disease was assigned for a period of 180 d. Clinical signs, plasma ACTH and alpha-MSH, the cortisol/creatinine urine ratio, and pituitary magnetic resonance imaging were assessed and compared at different time points. We recorded a significant reduction in plasma ACTH and alpha-MSH, and also in the cortisol/creatinine urine ratio, of the dogs treated with retinoic acid. Pituitary adenoma size was also significantly reduced at the end of retinoic acid treatment. Survival time and all the clinical signs evaluated showed an improvement in the retinoic-acid-treated dogs. No adverse events or signs of hepatotoxicity were observed, suggesting that the drug is not only effective but also safe. Retinoic acid treatment controls ACTH and cortisol hyperactivity and tumor size in dogs with ACTH-secreting tumors, leading to resolution of the clinical phenotype. This study highlights the possibility of using retinoic acid as a novel therapy in the treatment of ACTH-secreting tumors in humans with Cushing's disease.

Hypothalamus-pituitary-adrenal axis during Trypanosoma cruzi acute infection in mice.

Functional interactions between neuroendocrine and immune systems are mediated by similar ligands and receptors, which establish a bi-directional communication that is relevant for homeostasis. We investigated herein the hypothalamus-pituitary-adrenal (HPA) axis in mice acutely infected by Trypanosoma cruzi, the causative agent of Chagas' disease. Parasites were seen in the adrenal gland, whereas T. cruzi specific PCR gene amplification product was found in both adrenal and pituitary glands of infected mice. Histological and immunohistochemical analyses of pituitary and adrenal glands of infected animals revealed several alterations including vascular stasis, upregulation of the extracellular matrix proteins fibronectin and laminin, as well as T cell and macrophage infiltration. Functionally, we detected a decrease in CRH and an increase in corticosterone contents, in hypothalamus and serum respectively. In contrast, we did not find significant changes in the amounts of ACTH in sera of infected animals, whereas the serum levels of the glucocorticoid-stimulating cytokine, IL-6 (interleukin-6), were increased as compared to controls. When we analyzed the effects of T. cruzi in ACTH-producing AtT-20 cell line, infected cultures presented lower levels of ACTH and pro-opiomelanocortin production when compared to controls. In these cells we observed a strong phosphorylation of STAT-3, together with an increased synthesis of IL-6, suppressor of cytokine signaling 3 (SOCS-3) and inhibitor of activated STAT-3 (PIAS-3), which could explain the partial blockage of ACTH production. In conclusion, our data reveal that the HPA axis is altered during acute T. cruzi infection, suggesting direct and indirect influences of the parasite in the endocrine homeostasis.

Sonic hedgehog regulates CRH signal transduction in the adult pituitary.

Sonic hedgehog (Shh) is a signaling protein that binds to Patched and mediates its effects through Gli transcription factors. Shh is important in regulating survival and growth in both the embryo and the adult. It is known to be involved in pituitary development, but its role in the adult pituitary has not been investigated. Here, we show Shh and Gli1 immunoreactivity in adult human corticotroph cells. Administration of Shh (5 microg/ml) alone and in combination with corticotrophin-releasing hormone (CRH; 100 nM) in dispersed rat anterior pituitary and AtT-20 mouse corticotrophinoma cells increased corticotrophin (ACTH) secretion and pro-opiomelanocortin (POMC) promoter activity. Shh and CRH act additively in increasing CRH receptor 1 (CRH-R1). Unexpectedly, we found that CRH on its own increased Gli-dependent transcription, which in turn stimulated POMC transcription. Gli1 is necessary for CRH signaling, since knocking down Gli1 by RNA interference abolished the stimulatory effect of CRH on POMC. Taken together, our results demonstrate a new role for Shh and Gli1 in corticotroph function and provide a new link between Shh and CRH signaling pathways.