Proopiomelanocortin Gene Transcription Research Articles

OR11-05 High-throughput Screening Identifies TR4 Modulators For Potential Therapeutic Application

Abstract Disclosure: S. Khowal: None. D. Zhang: None. R. Damoiseaux: None. K. Javaherian: None. A.P. Heaney: None. Cushing Disease (CD), due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, leads to excess cortisol and is a life-threatening “orphan disease”. Although surgical removal can be effective, CD frequently recurs. Thereafter, repeat surgery, radiation therapy and/or bilateral adrenalectomy are not always successful and cause significant morbidity. Medical therapy has significantly improved but there is a clear unmet need for efficacious and safe therapies that target the pituitary corticotroph tumor itself to offer biochemical and tumor control. We previously demonstrated that the orphan testicular receptor 4 (TR4) regulates pro-opiomelanocortin gene transcription and ACTH secretion. Herein, we used the Flexi Vector system to identify TR4 modulators by fusing the TR4 LBD (380-615aa) to a 3’ GAL4 DBD to generate a chimeric fusion plasmid, pBIND-TR4-LBD. The pBIND-TR4-LBD construct and an exogenous luciferase reporter construct pGL4.35 (luc2P/9XGAL4UAS), containing 9 copies of a GAL4 responsive upstream activating sequence (UAS), were then transiently transfected into human embryonic kidney (HEK293) cells. Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. In a high throughput screen (HTS) of 27,705 chemicals, we identified several potential “hit” TR4 modulators that induced or inhibited TR4 LBD-directed GAL4UAS-directed luciferase expression by >3 -fold. Compounds from the HTS included ATP-sensitive K+ and calcium channel blockers, steroid hormones and modulators of cell proliferation and inflammation and several agonist and antagonist “hit” compounds induced TR4 >10 fold or inhibited TR4 by 90% respectively with minimal cell toxicity (<10%). In silico analysis of interactions between the TR4 LBD and these potential ligands indicated that the GLU-534 & ARG-565 may be crucial residues involved in TR4 ligand binding. Dose-response curves to calculate EC50 of these potential TR4 hit compounds and mode-of-action characterization are in progress. Our studies have identified several potent TR4 agonistic and antagonistic compounds and lay the framework for the discovery of safe and efficacious lead TR4 ligands that can be further advanced as potential drug therapies for CD and other diseases. Presentation: Friday, June 16, 2023

PMON149 Identification of TR4 Modulators for Treatment of Cushing Disease.

Abstract Cushing Disease (CD) is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that causes excess adrenal-derived cortisol. It is a life-threatening "orphan disease" with an annual health care cost >7-fold higher than average patients. Surgical removal is the current first-line therapy but the disease frequently recurs. Repeat surgery, radiation therapy and bilateral adrenalectomy are not always successful and associated with significant morbidity. Currently available drugs or those in clinical trials for CD do not target the pituitary corticotroph tumor itself and escape from control is common with long-term use. A clear unmet need for efficacious and safe therapies that offer biochemical and tumor control exists. We hypothesize that direct targeting of corticotroph tumors to modulate ACTH and in turn, glucocorticoid secretion is the optimal way to treat CD. We recently demonstrated that the orphan testicular receptor 4 (TR4, also known as NR2C2) is a potent regulator of hypothalamic-pituitary-adrenal (HPA) axis function and directly regulates pro-opiomelanocortin (POMC) gene transcription and ACTH secretion. To identify and characterize TR4 small molecule modulators, we used the Flexi Vector system to fuse the TR4 224-615aa LBD to the 3' GAL4 DBD to generate a chimeric fusion plasmid (pBIND-TR4-LBD) that constitutively expresses a Renilla luciferase serving as an internal reporter control. We then transiently transfected our chimeric pBIND-TR4-LBD construct into human embryonic kidney (HEK293) cells which express an exogenous reporter construct pGL4.35 (GloResponse, luc2P/9XGAL4UAS-HEK293 Cell Line, Promega) that is driven by multiple copies of a GAL4 responsive upstream activating sequence (UAS). Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. This dual-luciferase format allows us to detect compound cytotoxicity or off-target change in expression during chemical screening, greatly improving data quality. In a pilot drug screen of a LOPAC library and using robust z-score statistics (z-score>3), representing a luciferase to renilla induction > 3 standard deviations of baseline variability, we have identified several G-protein coupled ion channel regulators as potential "hit" TR4 modulators. These identified GPCRs regulate phosphatidylinositol and cyclic AMP effector systems, and activate several signaling pathway kinases. Mode-of-action and dose-response evaluation to calculate EC50 of these potential TR4 hit compounds is under investigation and lead compounds will be further validated using a POMC transactivation assay in murine and human corticotroph tumor cells. Our studies have begun to identify and rigorously validate compounds that efficiently and specifically abrogate TR4 actions to inhibit ACTH secretion and may lead to the discovery of safe and efficacious lead TR4 inhibitory compounds that can be further advanced as potential drug therapies for CD. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

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.

Regulation of pro-opiomelanocortin (POMC) gene transcription by interleukin-31 via early growth response 1 (EGR-1) in HaCaT keratinocytes.

Pro-opiomelanocortin (POMC) is a large precursor protein of and β-endorphin. POMC expressed in keratinocytes regulates various pathophysiological responses, such as pruritus in atopic dermatitis. Interleukin (IL)-31 is a T helper 2 (Th2)-derived cytokine that functions as a pruritogen, stimulating the sensory neurons in the skin. However, the regulatory mechanism underlying IL-31-induced POMC expression in keratinocytes remains largely unknown. Herein, using a 5'-serial deletion and site-specific mutation constructs of the regulatory region of POMC, we demonstrated that a putative EGR1-binding sequence (EBS) motif in POMC is required for its upregulation by IL-31 in HaCaT keratinocytes. Notably, EGR-1 directly interacted with the EBS motif in POMC. The ectopic expression of EGR-1 stimulated the POMC promoter activity, whereas the knockdown of EGR-1 expression by RNA interference reduced IL-31-induced POMC expression. Furthermore, we observed that three major mitogen-activated protein kinases, ERK, JNK, and p38 kinase, mediated IL-31-induced EGR-1 expression. In summary, our results suggest that EGR-1 trans-activates POMC in response to IL-31 stimulation in HaCaT keratinocytes.

Peptoid NPhe4 in AGRP-Based c[Pro1-Arg2-Phe3-Phe4-Xxx5-Ala6-Phe7-DPro8] Scaffolds Maintain Mouse MC4R Antagonist Potency.

The melanocortin receptors are involved in numerous physiological functions and are regulated by agonists derived from the proopiomelanocortin gene transcript and two endogenous antagonists, agouti and agouti-related protein (AGRP). The key binding and functional determinant of AGRP, an MC3R and MC4R antagonist, is an Arg-Phe-Phe tripeptide sequence located on an exposed hexapeptide (Arg-Phe-Phe-Asn-Ala-Phe) loop. It has previously been observed that cyclizing this sequence through a DPro-Pro motif (c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-DPro8]) resulted in a macrocyclic scaffold with MC4R antagonist activity, with increased MC4R potency when a diaminopropionic acid (Dap) residue is substituted at position 5. In this report, a series of 11 single-peptoid substitutions were performed in the AGRP-derived macrocycles. While most peptoid substitutions decreased MC4R antagonist potency, it was observed that NPhe4 (compounds 4 and 11) or NDab5 (diaminobutyric acid, compound 7) maintained MC4R antagonist potency. The NPhe4 substitutions also resulted in MC5R antagonist and inverse agonist activity equipotent to the parent scaffolds. These data may be used in the design of future MC4R and MC5R antagonist leads and probes that possess increased metabolic stability due to the presence of peptoid residues.

Single Nucleotide Polymorphisms in the Melanocortin His-Phe-Arg-Trp Sequences Decrease Tetrapeptide Potency and Efficacy.

The melanocortin receptors are stimulated by agonists (α-MSH, β-MSH, γ-MSH, and ACTH) processed from the proopiomelanocortin (POMC) gene transcript and possess a common His-Phe-Arg-Trp tetrapeptide sequence critical for receptor activation. Deficiency in POMC signaling in humans is associated with adrenal insufficiency, altered pigmentation, and rapid, early onset weight gain. Herein, 12 single nucleotide polymorphisms (SNPs) deposited into the Variation Viewer database within the His-Phe-Arg-Trp sequences of ACTH/α-MSH, β-MSH, and γ-MSH were substituted into tetrapeptide scaffolds to examine the in vitro signaling effects of these polymorphisms at the cloned melanocortin receptors. Every polymorphism decreased agonist potency and/or efficacy at the melanocortin receptors assayed, indicating that polymorphisms within the signaling sequence of POMC-derived agonists negatively impacts receptor activation. Future work to incorporate these substitutions into the full-length POMC agonists would confirm these findings, identifying new patient populations that might benefit from therapeutic regiments to treat POMC-deficient signaling.

Arg-Phe-Phe d-Amino Acid Stereochemistry Scan in the Macrocyclic Agouti-Related Protein Antagonist Scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro] Results in Unanticipated Melanocortin-1 Receptor Agonist Profiles.

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized β-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding d-isomer(s), generating a 14 compound library. While l-to-d inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.

Structure-Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor.

The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

A fragment of the Escherichia coli ClpB heat-shock protein is a micromolar melanocortin 1 receptor agonist.

The melanocortin system consists of five receptor subtypes (MC1-5R), endogenous agonists derived from the proopiomelanocortin gene transcript, and the antagonists agouti and agouti-related protein. The Escherichia coli heat shock protein ClpB has previously been described as an antigen mimetic to the endogenous melanocortin agonist α-MSH. Herein, we investigated if a fragment of the ClpB protein could directly signal through the melanocortin receptors. We synthesized a complementary fragment of the ClpB protein that partially aligned with α-MSH. Pharmacological assessment of this fragment resulted in no antagonist activity at the MC3R or the MC4R and no agonist activity at the MC4R. Partial receptor activation was observed for the MC3R and MC5R at 100 μM concentrations. This fragment was shown to be a full micromolar MC1R agonist and may serve as a template for future research into selective MC1R ligands.

Fetal alcohol exposure alters proopiomelanocortin gene expression and hypothalamic-pituitary-adrenal axis function via increasing MeCP2 expression in the hypothalamus.

Proopiomelanocortin (POMC) is a precursor gene of the neuropeptide β-endorphin in the hypothalamus and is known to regulate various physiological functions including stress response. Several recent reports showed that fetal alcohol exposure programs the hypothalamus to produce lower levels of POMC gene transcripts and to elevate the hypothalamic-pituitary-adrenal (HPA) axis response to stressful stimuli. We investigated the role of methyl CpG binding protein (MeCP2) in the effects of prenatal ethanol on POMC gene expression and hypothalamic-pituitary-adrenal (HPA) axis function. Pregnant Sprague Dawley rats were fed between GD 7 and 21 with a liquid diet containing 6.7% alcohol, pair-fed with isocaloric liquid diet, or fed ad libitum with rat chow, and their male offsprings were used at 60 days after birth in this study. Fetal alcohol exposure reduced the level of POMC mRNA, but increased the level of DNA methylation of this gene in the arcuate nucleus (ARC) of the hypothalamus where the POMC neuronal cell bodies are located. Fetal alcohol exposed rats showed a significant increase in MeCP2 protein levels in POMC cells, MeCP2 gene transcript levels as well as increased MeCP2 protein binding on the POMC promoter in the arcuate nucleus. Lentiviral delivery of MeCP2 shRNA into the third ventricle efficiently reduced MeCP2 expression and prevented the effect of prenatal ethanol on POMC gene expression in the arcuate nucleus. MeCP2-shRNA treatment also normalized the prenatal ethanol-induced increase in corticotropin releasing hormone (CRH) gene expression in the hypothalamus and elevated plasma adrenocorticotrophic hormone (ACTH) and corticosterone hormone responses to lipopolysaccharide (LPS) challenge. These results suggest that fetal alcohol programming of POMC gene may involve recruitment of MeCP2 on to the methylated promoter of the POMC gene to suppress POMC transcript levels and contribute to HPA axis dysregulation.

Modulation of proopiomelanocortin gene expression by ethanol in mouse anterior pituitary corticotrope tumor cell AtT20

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5–15mM concentrations of ethanol. After 15mM ethanol for 60 to 120min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60min ethanol at 5 to 15mM on the POMC PT levels was attenuated by 50μML-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

The Ets Factor Etv1 Interacts with Tpit Protein for Pituitary Pro-opiomelanocortin (POMC) Gene Transcription

Pro-opiomelanocortin (POMC) is expressed in two lineages of the pituitary, the anterior lobe corticotrophs and the intermediate lobe melanotrophs. POMC expression in these two lineages is highly dependent on the cell-restricted transcription factor Tpit. As Tpit intervenes relatively late in differentiation of those lineages, we have been searching for other transcription factors that may participate in their gene expression program. On the basis of similarity with the Tpit expression profile, we identified Ets variant gene 1 (Etv1/Er81) as a putative POMC transcription factor. Using Etv1-lacZ knockin mice, we describe preferential Etv1 expression in pituitary POMC cells and also in posterior lobe pituicytes. We further show that Etv1 enhances POMC transcription on its own and in synergy with Tpit. The Ets-binding site located within the Tpit/Pitx regulatory element is necessary for Etv1 activity in POMC-expressing AtT-20 cells but dispensable for synergy with Tpit. Etv1 and Tpit interact together in coimmunoprecipitation experiments. Furthermore, Etv1 is present at the POMC promoter, and siRNA-mediated knockdown of Etv1 in AtT-20 cells produces a significant decrease in POMC expression. Etv1 knockout pituitaries show normal POMC cell distribution and normal POMC mRNA abundance, suggesting compensation by other factors. The coordinate expression of Etv1 with POMC cell differentiation and its interaction with the highly cell-restricted Tpit factor indicate that Etv1 participates in a combinatorial code for pituitary cell-specific gene expression.

Involvement of Nuclear Factor-ĸB and Nurr-1 in Cytokine-Induced Transcription of Proopiomelanocortin Gene in AtT20 Corticotroph Cells

Objective: The precise mechanism whereby proinflammatory cytokines activate the hypothalamo-pituitary-adrenal axis is still unclear. We examined whether transcription factors nuclear factor (NF)-ĸB and Nurr-1 are involved in the cytokine-induced proopiomelanocortin (POMC) gene expression. Methods: The mouse corticotropinoma cell line AtT20 was treated with tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Real-time PCR, luciferase assay and Western blotting were conducted to assess the gene expression, promoter activity and protein expression in various conditions. Results: Intrinsic expression of NF-ĸB was confirmed by RT-PCR. An active component of NF-ĸB (p65) was upregulated in the nuclear fraction by both TNF-α and IL-1β treatment in a dose- and time-related manner. These cytokines potently stimulated the promoter activity of NF-ĸB and Nurr-1. We also found rapid upregulation of the Nurr-1 gene and protein after treatment with these cytokines. Cotreatment of the cells with either of the cytokines and corticotropin-releasing hormone resulted in additive effects. Cytokine-induced Nurr-1 transcription and Nurr-1 transcription induced by overexpression of NF-ĸB were both blunted by mutagenesis within the NF-ĸB responsive element, which implies that Nurr-1 upregulation specifically requires NF-ĸB binding to its own DNA-binding site. Proinflammatory cytokines exert positive effects on POMC gene expression, which were inhibited by pretreatment with a specific NF-ĸB inhibitor. Conclusion: These results together imply that Nurr-1 expression is a connecting point between neuroendocrine and immune systems in mediating cytokine-induced POMC gene expression.

Structure of the POMC promoter region in pituitary and extrapituitary ACTH producing tumors.

Proopiomelanocortin (POMC) is the common precursor of a variety of important endocrine peptides including ACTH. Transcription of the POMC gene is positively regulated by CRH through cAMP-responsive regions and is under negative feedback control by glucocorticoids which exert their inhibitory effect trough negative glucocorticoid responsive elements (nGRE). In vitro studies using the rat POMC promoter suggested that binding of the glucocorticoid receptor complex to a -63 bp binding site is correlated with repression of POMC gene transcription, and that specific mutations in this region abolish this effect. Impaired negative feedback regulation, though to a different degree, is a common feature of both corticotroph tumors (Cushing's disease) and extrapituitary ACTH producing tumors. We have analyzed the upstream promoter region of POMC gene from eleven patients with Cushing's disease, four of which had Nelson's syndrome, and from one patient with an ectopic ACTH syndrome secondary to a lung carcinoid for any possible mutations in the nGRE and/or cAMP-responsive sequences. DNA was purified from tumor tissue and was used as template for polymerase chain reaction (PCR). A segment between -371 and -19 bp of the POMC transcription start site was amplified and cloned into a plasmid vector. Sequencing was performed using the dideoxy chain termination procedure. Analysis of the 5'-flanking region revealed no defect in all tumors investigated. We conclude from our results that the defective glucocorticoid repression of POMC peptides production may be more likely due to aberrancies in other components of the complex transcriptional regulatory mechanism.

TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1beta by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1beta, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krüppel-associated box domain-containing zinc finger transcription factors. TIF1beta is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1beta is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1beta interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1beta enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1beta is recruited to the POMC promoter following CRH stimulation and that TIF1beta potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1beta and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1beta is an important coactivator of NGFI-B-dependent transcription.

Effects of orchidectomy on adaptation of arcuate neuropeptide Y, proopiomelanocortin, and cocaine- and amphetamine-related transcript gene profiles to recurring insulin-induced hypoglycemia in the male rat

Our studies show that recurring insulin-induced hypoglycemia (RIIH) diminishes neuronal activation in several key components of the central metabolic regulatory circuitry, including the hypothalamic arcuate nucleus (ARH), and that orchidectomy (ORDX) modifies this impact of RIIH on Fos protein expression in this and other select neural structures. The testicular hormone, testosterone, regulates the expression of ARH neuropeptide genes of characterized metabolic relevance, including neuropeptide Y (NPY) and proopiomelanocortin (POMC). We investigated the hypothesis that acute hypoglycemia-associated patterns of ARH NPY, POMC, and cocaine and amphetamine-related transcript (CART) gene transcription, and potential RIIH-associated adaptive modifications in these expression profiles are regulated by testes-dependent mechanisms. ARH tissue was micropunched from serial frozen brains sections obtained from sham-operated (SHAM) or bilaterally ORDX male rats after sc injection of one or four doses of neutral protamine Hagedorn insulin, over as many days, or vehicle alone, and analyzed by quantitative real-time RT-PCR. In SHAM rats, acute hypoglycemia increased ARH NPY mRNA; precedent hypoglycemia elevated baseline gene expression in this group, but suppressed transcription during RIIH. In ORDX rats, ARH NPY mRNA was decreased during acute hypoglycemia and after multiple exposures; however, gene expression was not further suppressed by RIIH. ARH POMC gene transcription was not modified by acute or recurring hypoglycemia in the SHAM group. ORDX caused a reduction in both basal and acute hypoglycemic patterns of POMC transcription, relative to the SHAM controls, but enhanced baseline and RIIH-associated patterns of gene expression. ARH CART transcripts were not altered by acute or recurring hypoglycemia in SHAM rats, whereas ORDX animals exhibited elevated CART gene expression during RIIH. These data show that acute and recurring hypoglycemia exert opposite effects on ARH NPY gene expression in testes-intact male rats, and that these transcriptional responses are abolished by ORDX. Hypoglycemia had no impact on POMC nor CART mRNA profiles in the SHAM group, but both genes were upregulated during RIIH in ORDX rats. Collectively, these results demonstrate that in the male rat, testes-dependent mechanisms underlie patterns of acclimated or unvarying reactivity to RIIH of the specific ARH metabolic neuropeptide genes evaluated here.

Evaluation of proopiomelanocortin mRNA in the peripheral blood from patients with Cushing’s syndrome of different origin

ACTH-dependent Cushing's syndrome is due to ACTH overproduction originating from a pituitary corticotroph adenoma (Cushing's disease) or from ectopic tumors (ectopic ACTH syndrome). Due to difficulties in the differential diagnosis between these two forms of hypercortisolism it would be important to have molecular tools able to discriminate the two conditions. It is known that proopiomelanocortin (POMC) gene transcription can originate messengers of different length. ACTHomas show the normal 1072 nucleotides (nt) transcript, whereas ectopic tumors seem to be associated with a longer mRNA form (1450 nt). In order to analyse the presence of different POMC transcripts, we extracted total RNA from peripheral lymphocytes of 10 patients with Cushing's disease, 10 with ectopic Cushing syndrome, and 20 controls as well as from pituitary tissues (2 ACTH-omas and a normal pituitary polyA+ sample). Northern blot analysis correctly revealed a 1072 nt mRNA molecule in pituitary ACTH-oma and in the normal pituitary polyA+ RNA samples, whereas neither this molecule nor other alternative transcripts were detected in blood samples from patients and controls. These data were confirmed by the more sensitive RT-PCR technique. This study further underlines the need for alternative approaches in the diagnosis of ACTH-dependent Cushing's syndrome.

Effects of Estradiol on Acute and Recurrent Insulin-Induced Hypoglycemia-Associated Patterns of Arcuate Neuropeptide Y, Proopiomelanocortin, and Cocaine- and Amphetamine-Related Transcript Gene Expression in the Ovariectomized Rat

The ovarian steroid hormone, estradiol, is one of several peripheral metabolic signal modulators that are integrated at the level of the arcuate nucleus of the hypothalamus (ARH), and is implicated in the control of ARH neuropeptides that maintain energy balance, including neuropeptide Y (NPY) and proopiomelanocortin (POMC). The present studies utilized quantitative real-time RT-PCR techniques to examine the hypothesis that estradiol regulates ARH NPY, POMC, and cocaine- and amphetamine-related transcript (CART) gene expression during acute insulin-induced hypoglycemia (IIH) and that adaptive modifications in transcriptional reactivity during recurring exposure are steroid dependent. ARH tissue was obtained by micropunch dissection from estradiol benzoate- and oil-implanted ovariectomized (OVX) rats that were treated by subcutaneous injection of one or four doses of the intermediate insulin formulation, Humulin NPH, over as many days, or vehicle alone. Our data show that in OVX plus estradiol benzoate and OVX plus oil groups, a single injection of insulin did not modify gene expression profiles, with the exception of acute hypoglycemic reduction of ARH NPY transcripts in the presence of estrogen. Prior exposure to daily hypoglycemia significantly diminished basal NPY and POMC mRNA levels in estradiol benzoate-, but not oil-implanted OVX rats, but elevated baseline CART transcripts in oil-treated animals. Recurring IIH enhanced ARH NPY gene expression relative to baseline, irrespective of the estradiol manipulation, but net tissue levels were greater in the absence of estrogen. In contrast, reexposure to hypoglycemia decreased POMC and CART gene transcription in estradiol benzoate- and oil-implanted OVX animals, respectively, relative to the single-dose groups. These studies show that estrogen modulates the impact of precedent exposure to IIH on basal and/or hypoglycemia-associated patterns of expression of ARH neuropeptide genes of characterized significance for energy homeostasis. The novel evidence for transcriptional acclimation of NPY, POMC, and CART to recurring IIH supports the possibility that adaptation of compensatory behavioral and physiological responses to acute versus chronic exposure to this metabolic stress may reflect neural regulatory mechanisms involving one or more neurotransmitters encoded by these genes.

Does p53 regulate skin pigmentation by controlling proopiomelanocortin gene transcription?

A recent issue of Cell highlights an intriguing, unexpected role for p53 as the central regulator of pigmentary responses in tanning and pathologic hyperpigmentation (Cui et al., 2007). Furthermore, in claiming to have provided biochemical and genetic evidence that the UV induction of intracutaneous proopiomelanocortin (POMC) (and its enzymatic cleavage products, a-MSH (melanocyte stimulating hormone) and b-endorphin) is directly controlled by p53, the authors propose that UV-induced tanning mainly follows a linear sequence of events that starts with a p53-mediated activation of the POMC promoter (Cui et al., 2007). The potential clinical significance and the biological implications of the reported findings and concepts necessitate further critical discussion of the experimental evidence, and the conclusions drawn from it, in the context of the available information on the subject. First, a significant part of the authors’ conclusion is based on animal experiments performed with C57BL ⁄6 mice (wild-type and p53 null) mice. However, POMCknockout mice on the same C57BL6 background are well known to show normal melanin pigment production (Slominski et al., 2005a). This observation may weaken the authors’ basic concept and also questions the validity of the proposed main regulatory axis for the suntan response and pathological hyperpigmentation (i.e. UV fi p53 fi POMC fi melanin pigmentation). This central issue requires serious explanation, and should have been carefully discussed by the authors. In our view, published evidence supports the hypothesis that it may not be POMC and its products but rather the melanocortin-1 receptor (MC1-R) that is the key regulator of pigmentation (at least in mice) (Slominski et al., 2004, 2005b). Moreover, in the absence of POMC-derived melanocortins, the MC1-R can regulate melanogenesis through an intrinsic, ligand-independent activity, or can be activated by ligands other than melanocortins (Sanchez-Mas et al., 2004; Slominski et al., 2005a,b). Furthermore, the authors do not appear to acknowledge that the regulation of melanocyte behavior, including melanogenesis control, is very heterogeneous and multi-directional; this further underscores the complexity of microenvironment – specific controls in pigmentation, which cannot be satisfactorily explained by an organization consisting of simple linear sequences (Nordlund et al., 2006; Slominski et al., 2004). On this background, we consider it more likely that p53 acts as one important coordinator (among others), but not as the main or sole regulator of pigmentation in the suntan response and pathological hyperpigmentation. Second, the authors provide evidence for direct induction of POMC transcription by p53, yet show only for a moderate increase of POMC precursor immunoreactivity (no evidence is presented that the selected immunoreactive band in their Figures 1 and 2 actually represents POMC). In this context, the surprising, highly unconventional sequence of reported events (e.g. p53 induction by UV is maximal by 3 h, POMC immunoreactivity reportedly peaks at 6 h, but its mRNA transcription at 24 h) requires explanation. Moreover, the authors fail to show conclusive evidence that the detected immunoreactivities (which, again, are rather unconventional: e.g. nuclear location of a-MSH in keratinocytes) indeed represent a-MSH or b-endorphin peptides, as claimed (confirmatory HPLC (high performance liquid chromatography) or mass spectrometry evaluations of representative extracts were not performed). Also, after transcription POMC mRNA is generated by splicing, which then is translated into a protein precursor of approximately 30 kDa. This precursor is further processed in a cell type-specific and tissue microenvironment-specific manner through complex, sequential post-translational modifications (including endoand exopeptidase cleavage, amidation and acetylation), which end with the regulated secretion of the final product (Slominski et al., 2000; Smith and Funder, 1988). The complexity of these events, in fact, renders it rather unlikely that p53 would directly regulate production and release of a-MSH or b-endorphin (however, indirect effects are quite possible).

Activation of AMP-activated protein kinase stimulates proopiomelanocortin gene transcription in AtT20 corticotroph cells

Starvation is known to activate the hypothalamo-pituitary-adrenal (HPA) axis, a representative antistress system in the living organism. In this study, we investigated in vitro whether activation of the AMP-activated protein kinase (AMPK), which is known to occur in intracellular energy depletion, influences the expression of POMC gene that encodes adrenocorticotropin. We first confirmed that each subunit of AMPK was expressed in the AtT20 corticotroph cell line. We then found that AICAR, a cell-permeable AMP analog and an activator of AMPK, potently stimulated the 5'-promoter activity of POMC gene in a dose-dependent manner. The effects were promoter specific because AICAR enhanced the AP1-mediated POMC promoter activities but did not influence other transcription factor-induced transcription. The effect of AICAR on POMC gene transcription was completely eliminated by specific AMPK inhibitor compound C or by dominant negative AMPK, whereas overexpression of constitutively active AMPK mimicked the effect of AICAR. Finally, experiments using specific kinase inhibitors suggested that the PI 3-kinase-mediated signaling pathway is at least partly involved in the effect. Our results suggest that intracellular energy depletion with the resultant activation of AMPK directly stimulates the HPA axis at the pituitary level by increasing the expression of POMC gene.