Proopiomelanocortin Transcription Research Articles

ODP335 Mutual Effects of Orexin and BMPs on Gonadotropin Expression by Mouse Gonadotrope LβT2 Cells

Abstract Orexins are neuropeptides that express primarily in the hypothalamus and are produced in two isoforms, orexin A and orexin B. There are two kinds of G protein coupled receptors, orexin type 1 (OX1R) and type 2 (OX2R) receptors. Orexin has been reported to have key roles on sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine organs, and orexin affects the regulation of the endocrine system. In our previous experiments, we have revealed the impact of orexins on anterior pituitary functions. For instance, we reported that orexin A plays an inhibitory role in prolactin production through the suppression of endogenous bone morphogenetic protein (BMP) activity in rat pituitary lactotrope GH3 cells. It was also reported that orexin A enhances pro-opiomelanocortin (POMC) transcription by upregulating corticotropin-releasing hormone (CRH) receptor signaling and by downregulating BMP-Smad signaling in mouse corticotope AtT20 cells. However, the effects of orexin on the endocrine function regarding gonadotrope cells remain unclear. We have recently uncovered that core clock genes and BMPs have mutual effects on luteinizing hormone (LH) expression in a phase-dependent manner by mouse gonadotrope LβT2 cells. In the present study, we investigated the effect of orexin on pituitary gonadotropin expression using LβT2 cells, which express OX1R and OX2R, by focusing on the functional involvement of BMP system and clock genes. It was revealed that orexin A stimulation increased LHβ and FSHβ mRNA expression in a concentration-responsive manner in the absence of GnRH, and interestingly, GnRH co-treatment further upregulated LHβ mRNA expression in LβT2 cells. Regarding the interrelationships between the signalings of orexin and BMPs, it was also revealed that orexin A pretreatment enhanced the BMP receptor signaling detected as the Smad1/5/9 phosphorylation, indicating that orexin enables to upregulate the BMP actions in LβT2 cells. In our previous studies, we reported that several BMP ligands such as BMP-6, -7 and 15 expressed in LβT2 cells can promote gonadotropin transcription, in which BMP-6 regulates GnRH-stimulated LH expression by modulating the sensitivity to somatostatin analogs. Based on the present results, it was implied that endogenous orexin can be functionally involved in the underlying mechanisms of gonadotropin expression. Collectively, orexin enhances gonadotropin expression by regulating BMP signaling in gonadotrope cells. Here, we will also discuss functional involvement of clock genes in the regulatory system of gonadotropin secretion induced by orexin and BMPs. Presentation: No date and time listed

302 Increased nociceptive thresholds associated with red hair are caused by reduced MC4R signaling

Humans and mice with natural red hair have elevated basal pain thresholds and an increased sensitivity to opioid analgesics. We investigated the mechanisms responsible for higher nociceptive thresholds in red-haired mice resulting from a loss of melanocortin 1 receptor (MC1R) function and found that the increased thresholds are melanocyte-dependent but melanin-independent. MC1R loss of function decreases melanocytic pro-opiomelanocortin transcription and systemic melanocyte stimulating hormone (MSH) levels in the plasma of red-haired (Mc1re/e) mice.

Effect of 3 NR3C1 Mutations in the Pathogenesis of Pituitary ACTH Adenoma.

Glucocorticoids act through the glucocorticoid receptor (GR) encoded by the nuclear receptor subfamily 3 group C member 1 (NR3C1) gene. This study aimed to examine the function of NR3C1 variants and their possible pathogenic role in Cushing disease (CD). Next-generation sequencing was conducted in 49 CD patients. Corticotroph tumor GR protein expression was examined by immunohistochemistry (IHC). Constructs harboring the 3 NR3C1-mutant and wild-type (WT) GR were transfected into the murine corticotropic adenoma cell line (AtT-20), and GR protein expression was quantified by Western blot. Translocation activity was assessed by immunofluorescence and effects of the GR mutants on corticotroph tumor proliferation, pro-opiomelanocortin (POMC) transcription, and ACTH secretion were tested. Clinical features were similar in patients harboring the NR3C1 mutations and WT GR. Recurrent adenomas showed higher GR IHC scores than nonrecurrent tumors. In vitro studies demonstrated that the p.R469X mutant generated a truncated GR protein, and the p.D590G and p.Y693D GR mutants resulted in lower GR expression. Dexamethasone (DEX) treatment of AtT-20 cells demonstrated decreased DEX-induced nuclear translocation, increased cell proliferation, and attenuated suppression of POMC transcription of 3 GR mutants. Interestingly, the p.R469X GR mutant resulted in increased murine corticotroph tumor ACTH secretion compared to WT GR. Our findings identify 3/49 (6.1%) consecutive human corticotroph tumors harboring GR mutations. Further findings demonstrate the role NR3C1 plays in CD pathogenesis and offer insights into a novel treatment approach in this patient subset.

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.

Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease.

ContextMechanisms underlying pituitary corticotroph adenoma adrenocorticotropin (ACTH) production are poorly understood, yet circulating ACTH levels closely correlate with adenoma phenotype and clinical outcomes.ObjectiveWe characterized the 5′ ends of proopiomelanocortin (POMC) gene transcripts, which encode the precursor polypeptide for ACTH, in order to investigate additional regulatory mechanisms of POMC gene transcription and ACTH production.MethodsWe examined 11 normal human pituitary tissues, 32 ACTH-secreting tumors, as well as 6 silent corticotroph adenomas (SCAs) that immunostain for but do not secrete ACTH.ResultsWe identified a novel regulatory region located near the intron 2/exon 3 junction in the human POMC gene, which functions as a second promoter and an enhancer. In vitro experiments demonstrated that CREB binds the second promoter and regulates its transcriptional activity. The second promoter is highly methylated in SCAs, partially demethylated in normal pituitary tissue, and highly demethylated in pituitary and ectopic ACTH-secreting tumors. In contrast, the first promoter is demethylated in all POMC-expressing cells and is highly demethylated only in pituitary ACTH-secreting tumors harboring the ubiquitin-specific protease 8 (USP8) mutation. Demethylation patterns of the second promoter correlate with clinical phenotypes of Cushing disease.ConclusionWe identified a second POMC promoter regulated by methylation status in ACTH-secreting pituitary tumors. Our findings open new avenues for elucidating subcellular regulation of the hypothalamic-pituitary-adrenal axis and suggest the second POMC promoter may be a target for therapeutic intervention to suppress excess ACTH production.

Possible roles of brain derived neurotrophic factor and corticotropin releasing hormone neurons in the nucleus of hippocampal commissure functioning within the avian neuroendocrine regulation of stress

Corticotropin releasing hormone (CRH) neurons located in the nucleus of hippocampal commissure (NHpC) have been proposed to be involved in the avian neuroendocrine regulation of stress and appeared to respond prior to CRH neurons in the hypothalamic paraventricular nucleus (PVN) when food deprivation stress was applied. Since the response of the NHpC was rapid and short-lived, was it regulated differentially from CRH neurons in the PVN? We, therefore, applied immobilization stress to test whether the NHpC response was stressor specific. Gene expression of CRH and stress-related genes in the NHpC, PVN, anterior pituitary (APit) as well as plasma corticosterone (CORT) were determined. Furthermore, brain derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR) were examined regarding their possible roles in the regulation of CRH neurons. Data showed that rapid activation of CRH mRNA in the NHpC occurred and preceded a slower gene activation in the PVN, upregulation of proopiomelanocortin (POMC) transcripts in the APit and significant increases of CORT concentrations. Results suggested BDNF’s role in negative feedback between CRH and CRHR1 in the NHpC and positive feedback between CRH and CRHR1 in the PVN. In the APit, V1bR activation appeared responsible for sustaining CORT release when stress persisted. Overall, data suggest that the NHpC functions as part of the HPA axis of birds and perhaps a comparable extra-hypothalamic structure occurs in other vertebrates. Lay Summary The nucleus of the hippocampal commissure, a structure outside of the hypothalamus, shows rapidly increased neural gene expression that appears to contribute to the early activation of the traditional hypothalamic-pituitary-adrenal (HPA) axis responsible for the production of stress hormones.

USP8 Somatic Mutations in Cushing’s Disease and Silent Corticotropinomas

Background: Somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene have been described in Cushing’s disease (CD). These mutations increase proopiomelanocortin transcription resulting in ACTH production and seem to correlate with somatostatin receptor type 5 (SST5) expression. Aims: Screen USP8 in patients with corticotropinomas and correlate USP8 mutational status with SST5 expression in CD. Methods: Tumor DNA was extracted and then exon 14 amplified by PCR. SST5 was assessed by immunohistochemistry (clone UMB4) and quantified multiplying the percentage of positive cells (0,0%; <10%,1;10-50%, 2; 51-80%, 3; >80%, 4) and intensity (mild, 1; moderate, 2; intense, 3), giving a score (IRS) from 0-12 with ≥ 6 considered high. Results: Among 59 patients, 38 had CD and 21 silent corticotropinomas. In CD, 13 (34.2%) patients had pathogenic mutations (6 had p.Ser719del; 5 had p.Pro720Arg and 2 had p.Pro720Gln). In the mutated CD group, all were women and had median age of 34.5 years (20-46). Median ACTH was 64.7pg/mL [(34.8-330.0), normal <46], urinary free cortisol (UFC) 435.0μg/24h [(87.0-1386.0), normal <100], cortisol after overnight 1mg dexamethasone suppression test (ODST) 17.4μg/dL [(5.0-48.7), normal <1.8], salivary cortisol (SC) 8.1μg/dL [(1.0-15.5), normal <0.35]. Median largest tumor size was 0.9 cm (0-1.9), ki-67 1.7 (0.2-10.0) and IRS 12 (1-12). In wild-type CD group, 19 (76.0%) were women and had median age was 35.0 years old (14-62). Median ACTH was 59.7 (39.0-137.0), UFC 305.8 (77.0-1302.0), cortisol after ODST 23.6 (10.0-33.3), SC 0.67 (0.27-1.28). Median largest tumor diameter 0.7cm (0-3.3), ki-67 1.8 (0.2-10) and IRS 4 (0-12). SC was higher in mutated group compared to wild-type (p=0.001) as well as IRS (p=0.009). In silent corticotropinomas, 2 (9.5%) had pathogenic mutations (1 p.Ser718Pro and 1 p.Pro720Arg): male, 36 years old, 3.2 cm tumor, Ki-67 4%, IRS 6; and female, 52 years old, 3.4 cm tumor, Ki-67 2.5%, IRS 12, respectively. One tumor had a variant not reported as pathogenic (p.Thr739Ala): male, 46 years old, 3.7 cm tumor, Ki-67 0.5%, IRS 0. USP8-wild-type silent corticotropinomas had IRS 0-2. Conclusion: One third of CD patients presented with somatic USP8 mutation. Similar to another study, about 10% of silent corticotropinomas also presented somatic USP8 mutation. Expression of SST5 was high in USP8-mutated CD and higher than wild-type group.

Orexin A Enhances Pro-Opiomelanocortin Transcription Regulated by BMP-4 in Mouse Corticotrope AtT20 Cells.

Orexin is expressed mainly in the hypothalamus and is known to activate the hypothalamic–pituitary–adrenal (HPA) axis that is involved in various stress responses and its resilience. However, the effects of orexin on the endocrine function of pituitary corticotrope cells remain unclear. In this study, we investigated the roles of orexin A in pro-opiomelanocortin (POMC) transcription using mouse corticotrope AtT20 cells, focusing on the bone morphogenetic protein (BMP) system expressed in the pituitary. Regarding the receptors for orexin, type 2 (OXR2) rather than type 1 (OX1R) receptor mRNA was predominantly expressed in AtT20 cells. It was found that orexin A treatment enhanced POMC expression, induced by corticotropin-releasing hormone (CRH) stimulation through upregulation of CRH receptor type-1 (CRHR1). Orexin A had no direct effect on the POMC transcription suppressed by BMP-4 treatment, whereas it suppressed Smad1/5/9 phosphorylation and Id-1 mRNA expression induced by BMP-4. It was further revealed that orexin A had no significant effect on the expression levels of type I and II BMP receptors but upregulated inhibitory Smad6/7 mRNA and protein levels in AtT20 cells. The results demonstrated that orexin A upregulated CRHR signaling and downregulated BMP-Smad signaling, leading to an enhancement of POMC transcription by corticotrope cells.

919 syrup inhibits ROS-mediated leptin-induced anorexia by activating PPARγ and improves gut flora abnormalities

BackgroundWomen with postpartum psychiatric disorders are prone to severe anorexia. Clinical studies have revealed the efficacy of 919 syrup, a traditional Chinese medicine mixture against postpartum illnesses, such as in regulating maternal mood and improving postpartum anorexia. AimThis study investigated the mechanisms through which 919 syrup improved anorexia induced by postpartum stress, focussing on the combined peroxisome proliferator-activated receptor gamma (PPARγ) and leptin signalling pathway, and its effects on the structure of the gut flora. MethodsMice were randomly divided into five groups—control group, immobilisation stressed (IS) group (normal saline), pioglitazone (Piog; western medicine control) group, 919 syrup low-dose (TJD; 13.5 g/kg) group, and 919 syrup high-dose (TJG; 27.0 g/kg) group. The control group was housed normally. The other groups received IS for 3 h daily for 21 days. The treatments were initiated following the first postnatal day and were administered by gastric gavage. All mice were sacrificed under anaesthesia on postnatal day 22. Blood, hypothalamus, stomach, and faecal specimens were collected. Gene and protein expression levels of components of the PPARγ–leptin signalling pathway in the serum, hypothalamus, and stomach were determined. Immunofluorescence staining for proopiomelanocortin (POMC), phosphorylated signal transducer and activator of transcription 3 (pSTAT3), and leptin was performed to observe their spatial distributions in the hypothalamus and stomach. 16s rRNA gene sequencing and bioinformatics analysis of fecal specimens were performed. ResultsAfter IS, postpartum mice showed significantly reduced appetite and body weight, accompanied by abnormalities in the structure of the gut flora. Treatment with 919 syrup (27.0 g/kg) downregulated malondialdehyde and upregulated catalase, glutathione peroxidase, and superoxide dismutase by activating PPARγ, thereby affecting the expression of leptin signalling pathway components (leptin, leptin receptor, pSTAT3, POMC, and cocaine and amphetamine-related transcript and neuropeptide Y), and modulated the gut flora in stressed mice. Conclusion919 syrup improved appetite in mice with postnatal stress by activating PPARγ to induce crosstalk with the leptin signalling pathway, this mechanism was similar to that of PPARγ agonists. 919 syrup also improved gut flora structure, and the changes in the relative abundances of the gut flora strongly correlated with the expression levels of PPARγ and leptin pathway components.

Regulation of the EGFR Pathway by HSP90 Is Involved in the Pathogenesis of Cushing's Disease.

PurposeTo investigate the role of heat-shock protein Hsp90 in adrenocorticotropic hormone (ACTH)-secreting cells, and to explore the potential clinical application of an inhibitor of Hsp90, 17-N-allylamino-17-demethoxygeldanamycin(17-AAG) in corticotropinomas [also known as “Cushing’s disease” (CD)].MethodsCulture of mouse pituitary tumor [AtT-20/D16v-F2 (ATCC® CRL-1795™)] cells and human pituitary ACTH-secreting tumor cells were employed. Hepatocellular carcinoma cell line (HLE) was used to evaluate EGFR inhibition by 17-AAG. Cell viability was evaluated using a commercial kit. The ACTH level was measured by a radioimmunoassay. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to measure expression of proopiomelanocortin (POMC) mRNA. Western blotting was done to measure protein levels.Results17-AAG suppressed the viability and proliferation, and promoted the apoptosis, of AtT-20/D16v-F2 cells. 17-AAG suppressed the synthesis and secretion of ACTH in AtT-20/D16v-F2 cells and down-regulated POMC transcription. 17-AAG acted in a similar pattern upon treatment with human pituitary ACTH-secreting tumor cells. Inhibition by 17-AAG was stronger in human pituitary ACTH-secreting tumor cells carrying the ubiquitin-specific protease-8 (USP8) mutant in comparison with cells carrying wild-type USP8.ConclusionsThe HSP90 inhibitor 17-AAG reduced the viability and secretory function of human pituitary ACTH-secreting tumor cells, and tumor cells carrying the USP8 mutant were more sensitive to 17-AAG than tumor cells carrying wild-type USP8. 17-AAG could be a potential treatment option for CD.

Acute acetate administration increases endogenous opioid levels in the human brain: A [11C]carfentanil molecular imaging study.

Introduction:A recent study has shown that acetate administration leads to a fourfold increase in the transcription of proopiomelanocortin (POMC) mRNA in the hypothalamus. POMC is cleaved to peptides, including β-endorphin, an endogenous opioid (EO) agonist that binds preferentially to the µ-opioid receptor (MOR). We hypothesised that an acetate challenge would increase the levels of EO in the human brain. We have previously demonstrated that increased EO release in the human brain can be detected using positron emission tomography (PET) with the selective MOR radioligand [11C]carfentanil. We used this approach to evaluate the effects of an acute acetate challenge on EO levels in the brain of healthy human volunteers.Methods:Seven volunteers each completed a baseline [11C]carfentanil PET scan followed by an administration of sodium acetate before a second [11C]carfentanil PET scan. Dynamic PET data were acquired over 90 minutes, and corrected for attenuation, scatter and subject motion. Regional [11C] carfentanil BPND values were then calculated using the simplified reference tissue model (with the occipital grey matter as the reference region). Change in regional EO concentration was evaluated as the change in [11C]carfentanil BPND following acetate administration.Results:Following sodium acetate administration, 2.5–6.5% reductions in [11C]carfentanil regional BPND were seen, with statistical significance reached in the cerebellum, temporal lobe, orbitofrontal cortex, striatum and thalamus.Conclusions:We have demonstrated that an acute acetate challenge has the potential to increase EO release in the human brain, providing a plausible mechanism of the central effects of acetate on appetite in humans.

Exome Sequencing Identifies Genes and Gene Sets Contributing to Severe Childhood Obesity, Linking PHIP Variants to Repressed POMC Transcription

SummaryObesity is genetically heterogeneous with monogenic and complex polygenic forms. Using exome and targeted sequencing in 2,737 severely obese cases and 6,704 controls, we identified three genes (PHIP, DGKI, and ZMYM4) with an excess burden of very rare predicted deleterious variants in cases. In cells, we found that nuclear PHIP (pleckstrin homology domain interacting protein) directly enhances transcription of pro-opiomelanocortin (POMC), a neuropeptide that suppresses appetite. Obesity-associated PHIP variants repressed POMC transcription. Our demonstration that PHIP is involved in human energy homeostasis through transcriptional regulation of central melanocortin signaling has potential diagnostic and therapeutic implications for patients with obesity and developmental delay. Additionally, we found an excess burden of predicted deleterious variants involving genes nearest to loci from obesity genome-wide association studies. Genes and gene sets influencing obesity with variable penetrance provide compelling evidence for a continuum of causality in the genetic architecture of obesity, and explain some of its missing heritability.

Nuclear Receptors as Regulators of Pituitary Corticotroph Pro-Opiomelanocortin Transcription.

The hypothalamic–pituitary–adrenal (HPA) axis plays a critical role in adaptive stress responses and maintaining organism homeostasis. The pituitary corticotroph is the central player in the HPA axis and is regulated by a plethora of hormonal and stress related factors that synergistically interact to activate and temper pro-opiomelanocortin (POMC) transcription, to either increase or decrease adrenocorticotropic hormone (ACTH) production and secretion as needed. Nuclear receptors are a family of highly conserved transcription factors that can also be induced by various physiologic signals, and they mediate their responses via multiple targets to regulate metabolism and homeostasis. In this review, we summarize the modulatory roles of nuclear receptors on pituitary corticotroph cell POMC transcription, describe the unique and complex role these factors play in hypothalamic–pituitary–adrenal axis (HPA) regulation and discuss potential therapeutic targets in disease states.

Exome Sequencing Identifies Multiple Genes and Gene-Sets Contributing to Severe Childhood Obesity

Obesity is genetically heterogeneous with monogenic and complex polygenic forms. Using exome sequencing followed by targeted sequencing in 2,737 severely obese cases and 6,704 controls, we identified three genes (PHIP, DGKI, ZMYM4) with an excess burden in cases of variants (MAF<0.025%) predicted to affect function. In cells, we found that nuclear PHIP (Pleckstrin Homology domain Interacting Protein) directly enhances transcription of Pro-opiomelanocortin (POMC), a neuropeptide which suppresses appetite. Obesity-associated PHIP variants repressed POMC transcription. We found an excess burden of in silico deleterious variants (MAF<0.025%) in genes nearest to loci from obesity genome-wide association studies and genes intolerant to loss-of-function, in gene-set analyses. The discovery of genes and gene-sets influencing obesity in a non-fully penetrant manner provides mechanistic insights and has diagnostic and therapeutic implications.

Tonicity-responsive enhancer binding protein (TonEBP) regulates TNF-α-induced hypothalamic inflammation.

Tonicity-responsive enhancer binding protein (TonEBP) is a widely expressed transcription factor and is important in the regulation of inflammatory cytokines. Here, we have identified TonEBP expression in the hypothalamus, which is particularly high in proopiomelanocortin (POMC) neurons. TonEBP overexpression stimulates POMC transcription, and TonEBP haploinsufficiency in TonEBP (+/-) mice results in a decrease in hypothalamic POMC expression. TonEBP (+/-) mice show reduced sickness responses, which include anorexia and hyperthermia, that are initially induced by tumor necrosis factor (TNF)-α. TonEBP (+/-) mice also show lower levels of TNF-α-induced hypothalamic expression of POMC and pro-inflammatory cytokines. These results suggest that TonEBP is an important molecular regulator in the development of inflammatory sickness responses through the control of POMC and pro-inflammatory cytokine expression in the hypothalamus.

TSP-1 is downregulated and inversely correlates with miR-449c expression in Cushing's disease.

The pathogenesis of Cushing's disease, which is caused by pituitary corticotroph adenoma, remains to be studied. Secreted angioinhibitory factor thrombospondin‐1 (TSP‐1) is an adhesive glycoprotein that mediates cell‐to‐cell and cell‐to‐matrix interactions and is associated with platelet aggregation, angiogenesis and tumorigenesis. We have found that the expression of TSP‐1 is significantly lower in human pituitary corticotroph tumours compared with normal adenohypophysis. This study aims to elucidate the role of TSP‐1 in regulating the tumour function of pituitary adenomas. Forced overexpression of TSP‐1 in a murine AtT20 pituitary corticotroph tumour cell line decreased corticotroph precursor hormone proopiomelanocortin (POMC) transcription and adrenocorticotropic hormone (ACTH) secretion. Functional studies showed that TSP‐1 overexpression in pituitary adenoma cells suppressed proliferation, migration and invasion. We have demonstrated that TSP‐1 is a direct target of miR‐449c. Further study showed that miR‐449c activity enhanced tumorigenesis by directly inhibiting TSP‐1 expression. Low expression of lncTHBS1, along with low expression of TSP‐1, was associated with the high expression of miR‐449c in Cushing's disease patients. Furthermore, RNA‐immunoprecipitation associates miR‐449c with lncTHBS1 suggesting that lncTHBS1 might be a negative regulator of miR‐449c. Taken together, this study has demonstrated that lncTHBS1 might function as competing endogenous RNA for miR‐449c, which could suppress the development of Cushing's disease.

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.

SAT-156 Sex-Specific, Diet-Induced Metabolic Dysfunction in F1 Progeny as a Consequence of Maternal Adolescent Preconception Opioid Exposure

Adverse metabolic conditions (obesity and type-2 diabetes) continue to be prevalent in developed nations. While the etiology of these diseases is multifactorial (i.e., genetic, environmental), parental exposures/experiences can also impart unfavorable metabolic phenotypes in future non-exposed progeny. We are currently in the midst of an opioid epidemic, with adolescents representing a growing population now exposed to this class of drugs. Endogenous opioids regulate reward- and motivation- related behaviors and chronic exposure to opioids significantly alter the brain’s reward system, an effect that can be transmitted across multiple generations. Indeed, data from animal models demonstrate that adolescent opioid exposure in females can impact their future progeny, even when that exposure occurs prior to conception. Effects observed in the offspring of morphine exposed females (MORF1) when compared to the offspring of saline exposed females (SALF1) include increased transcription of proopiomelanocortin (POMC) within the hypothalamic arcuate nucleus (ARC) of MORF1 offspring. POMC is differentially processed into several bioactive neuropeptides that play key roles in homeostatic and adaptive mechanisms. For example, β- endorphin (β-EP) and α- melanocyte stimulating hormone (α-MSH) regulate both reward/motivation and metabolic pathways, thus influencing reward liability as well as energy balance. Utilizing a preclinical model of preconception female adolescent opioid exposure, the current study demonstrates that when fed an obesogenic diet for 6-weeks, MORF1 offspring sex-specifically develop several deleterious metabolic effects; they consume more food, gain more weight, and develop fasting-induced hyperglycemia, hyperinsulinemia, and hypercorticosteronemia. MORF1 effects are associated with diet-specific alterations in ARC expression of neuropeptides known to regulate energy homeostasis. Further, while analysis across the POMC locus revealed no differences related to DNA methylation status in F1 male offspring at baseline, RNA sequencing of ARC tissue revealed an overall down-regulation of genes associated with synaptic plasticity in MORF1 males maintained on an obesogenic diet. Moreover, this transcriptional effect is coupled with an upregulation of inflammatory transcriptional networks. Thus, females adolescently exposed to morphine, conferred innate changes to the transcriptional response of the ARC to an obesogenic diet in the next generation. Such an effect induces the development of features suggestive of the emergence of metabolic syndrome in F1 offspring. Collectively, these findings suggest a potential confluence of two major global health issues (the opioid epidemic and metabolic syndrome) and provides evidence that maternal indices of opioid exposure can adversely influence metabolic predisposition in future progeny.

Epigenetic alterations of the POMC promoter in tobacco dependence

Impaired regulation of the hypothalamic-pituitary-adrenal (HPA) axis is substantially involved in several psychiatric disorders. Smoking interferes with HPA axis by activating proopiomelanocortin (POMC) neurons and thus stimulating the expression of POMC. The POMC transcript is processed into several peptide hormones, such as adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH), that play a role in stress response and weight control. In alcohol dependence, POMC promoter methylation is associated with craving. Here, we describe evidence of altered POMC promoter methylation in smoking. To determine how tobacco dependence and its withdrawal affect POMC promoter-specific DNA methylation, we assessed blood samples of 36 tobacco dependent individuals at day 1, 7 and 14 of withdrawal compared to 41 healthy controls using direct bisulfite sequencing. We found that POMC promoter methylation is significantly higher in smokers than in non-smokers. Moreover, this methylation difference does not readapt within 14 days of abstinence. We offer two explanatory models: Smokers could have a higher methylation state before the onset of smoking and this premorbid status might be acquired by environmental factors in early life. Alternatively, smoking may activate POMC neurons and its protein expression. Therefore, increasing methylation status of its promoter might be an adjustment to keep homeostasis. In either way, altered POMC methylation in smokers seems to indicate an adaptation of stress signaling, thereby potentially serving as a marker for stress-related functions that support the addiction.

New Insights in Cushing Disease Treatment With Focus on a Derivative of Vitamin A.

Cushing’s disease (CD) is an endocrine disorder originated by a corticotroph tumor. It is linked with high mortality and morbidity due to chronic hypercortisolism. Treatment goals are to control cortisol excess and achieve long-term remission, therefore, reducing both complications and patient’s mortality. First-line of treatment for CD is pituitary’s surgery. However, 30% of patients who undergo surgery experience recurrence in long-term follow-up. Persistent or recurrent CD demands second-line treatments, such as pituitary radiotherapy, adrenal surgery, and/or pharmacological therapy. The latter plays a key role in cortisol excess control. Its targets are inhibition of adrenocorticotropic hormone (ACTH) production, inhibition of adrenal steroidogenesis, or antagonism of cortisol action at its peripheral receptor. Retinoic acid (RA) is a metabolic product of vitamin A (retinol) and has been studied for its antiproliferative effects on corticotroph tumor cells. It has been shown that this drug regulates the expression of pro-opiomelanocortin (POMC), ACTH secretion, and tumor growth in corticotroph tumor mouse cell lines and in the nude mice experimental model, via inhibition of POMC transcription. It has been shown to result in tumor reduction, normalization of cortisol levels and clinical improvement in dogs treated with RA for 6 months. The orphan nuclear receptor COUP-TFI is expressed in normal corticotroph cells, but not in corticotroph tumoral cells, and inhibits RA pathways. A first clinical human study demonstrated clinical and biochemical effectiveness in 5/7 patients treated with RA for a period of up to 12 months. In a recent second clinical trial, 25% of 16 patients achieved eucortisolemia, and all achieved a cortisol reduction after 6- to 12-month treatment. The goal of this review is to discuss in the context of the available and future pharmacological treatments of CD, RA mechanisms of action on corticotroph tumor cells, and future perspectives, focusing on potential clinical implementation.