A basal actinopterygian melanocortin receptor: Molecular and functional characterization of an Mc2r ortholog from the Senegal bichir (Polypterus senegalus).

In bony vertebrates, melanocortin 2 receptor (Mc2r) specifically binds adrenocorticotropic hormone (ACTH) and is responsible for mediating anterior pituitary signaling that stimulates corticosteroid production in the adrenal gland/interrenal cells. In bony fishes Mc2r requires the chaperoning of an accessory protein (Mrap1) to traffic to the membrane surface and bind ACTH. Here, we evaluated the structure and pharmacological properties of Mc2r from the Senegal bichir (Polypterus senegalus), which represents the most basal bony fish from which an Mc2r has been pharmacologically studied to date. In our experiments, cDNA constructs of the Mc2r from the Senegal bichir (sbMc2r) and various vertebrate Mrap1s were heterologously co-expressed in Chinese hamster ovary (CHO) cells, stimulated by ACTH or melanocyte-stimulating hormone (α-MSH) ligands, and assessed using a luciferase reporter gene assay. When expressed without an Mrap1, sbMc2r was not activated by ACTH. When co-expressed with Mrap1 from either chicken (Gallus gallus) or bowfin (Amia calva), sbMc2r could be activated in a dose-dependent manner by ACTH, but not α-MSH. Co-expression of sbMrap2 with sbMc2r resulted in no detectable activation of the receptor. Collectively, these results demonstrate that sbMc2r has pharmacological properties similar to those of Mc2rs of later-evolved bony fishes, such as Mrap1 dependence and ACTH selectivity, indicating that these qualities of Mc2r function are ancestral to all bony fish Mc2rs.

Identification of genes associated with human-canine communication in canine evolution.

The dog (Canis familiaris) was the first domesticated animal and hundreds of breeds exist today. During domestication, dogs experienced strong selection for temperament, behaviour, and cognitive ability. However, the genetic basis of these abilities is not well-understood. We focused on ancient dog breeds to investigate breed-related differences in social cognitive abilities. In a problem-solving task, ancient breeds showed a lower tendency to look back at humans than other European breeds. In a two-way object choice task, they showed no differences in correct response rate or ability to read human communicative gestures. We examined gene polymorphisms in oxytocin, oxytocin receptor, melanocortin 2 receptor, and a Williams-Beuren syndrome-related gene (WBSCR17), as candidate genes of dog domestication. The single-nucleotide polymorphisms on melanocortin 2 receptor were related to both tasks, while other polymorphisms were associated with the unsolvable task. This indicates that glucocorticoid functions are involved in the cognitive skills acquired during dog domestication.

A variant in the 3'-untranslated region of the MC2R gene decreases the risk of schizophrenia in a female Han Chinese population.

OBJECTIVE: Schizophrenia is a complex mental disorder with high heritability. The hypothalamic-pituitary-adrenal (HPA) axis, which is the stress system of the neuroendocrine system, is considered to impact psychotic disorders. We hypothesized that polymorphisms of HPA axis genes might be involved in the development of schizophrenia. METHODS: A case-control study comprising 234 patients with schizophrenia and 399 matched healthy controls was conducted to investigate the association between the human melanocortin 2 receptor (MC2R) gene and schizophrenia risk. Seven tag single nucleotide polymorphisms (SNPs) (rs16941303, rs16941314, rs2186944, rs28926188, rs7230126, rs948322, and rs948331) of MC2R were genotyped by direct sequencing. RESULTS: No significant associations were observed between any of the alleles, genotypes, or haplotypes examined within the MC2R gene and the risk of schizophrenia in the total group or in subgroups stratified by smoking or alcoholism. However, a subgroup analysis stratified by sex revealed that under the additive model, the C allele of the MC2R rs948331 SNP significantly decreased the risk of schizophrenia in females (odds ratio=0.18). CONCLUSION: The C allele of the MC2R rs948331 locus may be a protective factor, reducing the risk of schizophrenia in the female Han Chinese population.

Aldosterone-Regulating Receptors and Aldosterone-Driver Somatic Mutations.

Background: Somatic gene mutations that facilitate inappropriate intracellular calcium entrance have been identified in most aldosterone-producing adenomas (APAs). Studies suggest that angiotensin II and adrenocorticotropic hormone (ACTH) augment aldosterone production from APAs. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Objective: To analyze the transcript expression of type 1 angiotensin II receptors (AGTR1), ACTH receptors (MC2R), and melanocortin 2 receptor accessory protein (MRAP) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from APAs with mutations in: KCNJ5 (n = 14), ATP1A1 (n = 14), CACNA1D (n = 14), and ATP2B3 (n = 5), and from normal adjacent adrenal tissue (n = 45). Transcript expression of MC2R, MRAP, AGTR1, aldosterone synthase (CYP11B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11ß-hydroxylase (CYP11B1) were quantified using quantitative RT-PCR and normalized to ß-actin. Results: Compared to adjacent normal adrenal tissue, APAs had higher transcript levels of CYP11B2 (2,216.4 [1,112.0, 2,813.5]-fold, p < 0.001), MC2R (2.88 [2.00, 4.52]-fold, p < 0.001), and AGTR1 (1.80 [1.02, 2.80]-fold, p < 0.001]), and lower transcript levels of MRAP, CYP17A1, and CYP11B1 (0.28-0.36, p < 0.001 for all). MC2R and CYP11B2 transcripts were lower in APAs with KCNJ5 vs. other mutations (p < 0.01 for both). MC2R expression correlated positively with that of AGTR1 in APAs harboring KCNJ5 and CACNA1D mutations, and with MRAP expression in APAs harboring ATPase mutations. Conclusions: While MC2R and AGTR1 are expressed in all APAs, differences were observed based on the underlying aldosterone-driver somatic mutations. In tandem, our findings suggest that APAs with ATPase-mutations are more responsive to ACTH than KCNJ5-mutated APAs.

Melanocortin 2, 3 and 4 Receptor Gene Expressions are Downregulated in CD8+ T Cytotoxic Lymphocytes and CD19+ B Lymphocytes in Rheumatoid Arthritis Responding to TNF-α Inhibition.

Melanocortin signalling in leucocyte subsets elicits anti-inflammatory and immune tolerance inducing effects in animal experimental inflammation. In man, however, the effects of melanocortin signalling in inflammatory conditions have scarcely been examined. We explored the differential reactions of melanocortin 1-5 receptors (MC1-5R) gene expressions in pathogenetic leucocyte subsets in rheumatoid arthritis (RA) to treatment with TNF-α inhibitor adalimumab. Seven patients with active RA donated blood at start and at 3-month treatment. CD4+ T helper (h) lymphocytes (ly), CD8+ T cytotoxic (c) ly, CD19+ B ly and CD14+ monocytes were isolated, using immunomagnetic beads, total RNA extracted and reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed. Fold changes in MC1-5R, Th1-, inflammatory- and regulatory cytokine gene expressions were assessed for correlation. Six patients responded to adalimumab treatment, while one patient was non-responder. In all lymphocyte subtypes, MC1-5R gene expressions decreased in responders and increased in the non-responder. In responders, decrease in MC2R, MC3R and MC4R gene expressions in CD8+ Tc and CD19+ B ly was significant. Fold change in MC1-5R and IFNγ gene expressions correlated significantly in CD8+ Tc ly, while fold change in MC1R, MC3R and MC5R and IL-1ß gene expressions correlated significantly in CD4+ Th ly. Our results show regulation of MC2R, MC3R and MC4R gene expressions in CD8+ Tc ly and CD19+ B ly. The correlations between fold change in different MCRs and disease driving cytokine gene expressions in CD8+ Tc ly and CD4+ Th ly point at a central immune modulating function of the melanocortin system in RA.

Association of estradiol on expression of melanocortin receptors and their accessory proteins in the liver of chicken (Gallus gallus).

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R-MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17ß-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period.

Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex.

We have shown that fetal adrenal fetal zone (FZ) volume and serum dehydroepiandrosterone sulfate (DHAS) levels were increased, whereas definitive and transitional zone (DZ/TZ) volume was unaltered, in baboons in which estrogen levels were suppressed by the administration of the aromatase inhibitor letrozole. The interaction of the melanocortin 2 receptor (MC2R) with its accessory protein (MRAP) is essential for trafficking MC2R to the adrenal cell surface for binding to ACTH. The present study determined whether the estrogen-dependent regulation of fetal adrenocortical development is mediated by ACTH and/or expression/interaction of MC2R and MRAP. Fetal pituitary proopiomelanocortin mRNA and plasma ACTH levels and fetal adrenal MC2R-MRAP interaction were assessed in baboons in which estrogen was suppressed/restored by letrozole/letrozole plus estradiol administration during the second half of gestation. Although fetal pituitary proopiomelanocortin and plasma ACTH levels and fetal adrenal MC2R and MRAP protein levels were unaltered, MC2R-MRAP interaction was 2-fold greater (P < .05) in the DZ/TZ in letrozole-treated baboons than in untreated animals and restored by letrozole plus estradiol treatment. We propose that the increasing levels of estradiol with advancing pregnancy suppress interaction of MC2R with MRAP, thereby diminishing MC2R movement to the cell membrane in the DZ/TZ. This would be expected to reduce progenitor cell proliferation in the DZ and migration to the FZ, thereby restraining FZ growth and DHAS production to maintain fetal adrenal DHAS and placental estradiol levels in a physiological range late in gestation.

Exome genotyping arrays to identify rare and low frequency variants associated with epithelial ovarian cancer risk.

Rare and low frequency variants are not well covered in most germline genotyping arrays and are understudied in relation to epithelial ovarian cancer (EOC) risk. To address this gap, we used genotyping arrays targeting rarer protein-coding variation in 8,165 EOC cases and 11,619 controls from the international Ovarian Cancer Association Consortium (OCAC). Pooled association analyses were conducted at the variant and gene level for 98,543 variants directly genotyped through two exome genotyping projects. Only common variants that represent or are in strong linkage disequilibrium (LD) with previously-identified signals at established loci reached traditional thresholds for exome-wide significance (P < 5.0 × 10 - 7). One of the most significant signals (Pall histologies = 1.01 × 10 - 13;Pserous = 3.54 × 10 - 14) occurred at 3q25.31 for rs62273959, a missense variant mapping to the LEKR1 gene that is in LD (r2 = 0.90) with a previously identified 'best hit' (rs7651446) mapping to an intron of TIPARP. Suggestive associations (5.0 × 10 - 5 > P≥5.0 ×10 - 7) were detected for rare and low-frequency variants at 16 novel loci. Four rare missense variants were identified (ACTBL2 rs73757391 (5q11.2), BTD rs200337373 (3p25.1), KRT13 rs150321809 (17q21.2) and MC2R rs104894658 (18p11.21)), but only MC2R rs104894668 had a large effect size (OR = 9.66). Genes most strongly associated with EOC risk included ACTBL2 (PAML = 3.23 × 10 - 5; PSKAT-o = 9.23 × 10 - 4) and KRT13 (PAML = 1.67 × 10 - 4; PSKAT-o = 1.07 × 10 - 5), reaffirming variant-level analysis. In summary, this large study identified several rare and low-frequency variants and genes that may contribute to EOC susceptibility, albeit with possible small effects. Future studies that integrate epidemiology, sequencing, and functional assays are needed to further unravel the unexplained heritability and biology of this disease.

60 YEARS OF POMC: Adrenal and extra-adrenal functions of ACTH.

The pituitary adrenocorticotropic hormone (ACTH) plays a pivotal role in homeostasis and stress response and is thus the major component of the hypothalamo-pituitary-adrenal axis. After a brief summary of ACTH production from proopiomelanocortin (POMC) and on ACTH receptor properties, the first part of the review covers the role of ACTH in steroidogenesis and steroid secretion. We highlight the mechanisms explaining the differential acute vs chronic effects of ACTH on aldosterone and glucocorticoid secretion. The second part summarizes the effects of ACTH on adrenal growth, addressing its role as either a mitogenic or a differentiating factor. We then review the mechanisms involved in steroid secretion, from the classical Cyclic adenosine monophosphate second messenger system to various signaling cascades. We also consider how the interaction between the extracellular matrix and the cytoskeleton may trigger activation of signaling platforms potentially stimulating or repressing the steroidogenic potency of ACTH. Finally, we consider the extra-adrenal actions of ACTH, in particular its role in differentiation in a variety of cell types, in addition to its known lipolytic effects on adipocytes. In each section, we endeavor to correlate basic mechanisms of ACTH function with the pathological consequences of ACTH signaling deficiency and of overproduction of ACTH.

Clinical and Steroidogenic Characteristics of Aldosterone-Producing Adenomas With ATPase or CACNA1D Gene Mutations.

OBJECT: This comparative study clarified the clinical characteristics and in vitro steroidogenic activities of aldosterone-producing adenomas (APAs) harboring ATPase or CACNA1D gene mutations. DESIGN AND PATIENTS: Genetic testing was performed on 159 unilateral APAs. Somatic ATPase and CACNA1D gene mutations were analyzed in 42 APA tissues without KCNJ5 gene mutations. RESULTS: ATP1A1, ATP2B3, and CACNA1D mutations were detected in one, four, and four patients, respectively. Compared with patients without KCNJ5, ATPase, or CACNA1D mutations (wild type), ATPase mutations tended to have more severe hyperaldosteronism and smaller tumors; those with CACNA1D mutations had clinical characteristics and tumor sizes similar to those with wild-type genes. APAs with ATPase mutations were composed mainly of compact eosinophilic tumor cells, whereas CACNA1D mutations resulted in predominantly clear tumor cells. Aldosterone production in APA cells with ATP2B3 mutations were more responsive to dibutyryl cAMP, whereas those with CACNA1D mutations were more responsive to adrenocorticotropic hormone than the wild-type cells. CONCLUSION: APAs with ATPase mutations demonstrated a potentially severe primary aldosteronism phenotype, whereas those with CACNA1D mutations displayed characteristics similar to wild-type APAs. The status of stimulated aldosterone production was also different according to the cell types, suggesting that the regulatory effects of adrenocorticotropic hormone on aldosterone synthesis could possibly vary according to the intracellular signaling involved in hormone production.

Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality.

OBJECTIVE: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. METHODS/RESULTS: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R(-/-)) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R(+/-) mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R(+/-), but not of MC2R(-/-), mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R(+/-), but not of MC2R(-/-), mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. CONCLUSION: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

Melanocortins contribute to sequential differentiation and enucleation of human erythroblasts via melanocortin receptors 1, 2 and 5.

In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.

Abundance of DLK1, differential expression of CYP11B1, CYP21A2 and MC2R, and lack of INSL3 distinguish testicular adrenal rest tumours from Leydig cell tumours.

OBJECTIVE: Testicular adrenal rest tumours (TARTs) are a common finding in patients with congenital adrenal hyperplasia (CAH). These tumours constitute a diagnostic and management conundrum and may lead to infertility. TART cells share many functional and morphological similarities with Leydig cells (LCs), and masses consisting of such cells are occasionally misclassified as malignant testicular tumours, which may lead to erroneous orchiectomy in these patients. DESIGN: In this study, we aimed to investigate the potential of LC developmental markers and adrenal steroidogenic markers in the differential diagnosis of TARTs and malignant LC tumours (LCTs). METHODS: We investigated mRNA and protein expression of testicular steroidogenic enzymes; CYP11A1 and HSD3B1/2, markers of adrenal steroidogenesis; CYP11B1, CYP21A2 and ACTH receptor/melanocortin 2 receptor (MC2R), and markers of LC maturation; and delta-like 1 homolog (DLK1) and insulin-like 3 (INSL3) in testicular biopsies with TART, orchiectomy specimens with LCTs and samples from human fetal adrenals. RESULTS: Expression of testicular steroidogenic enzymes was observed in all specimens. All investigated adrenal steroidogenic markers were expressed in TART, and weak reactions for CYP11B1 and MC2R were observed at the protein level in LTCs. TART and fetal adrenals had identical expression profiles. DLK1 was highly expressed and INSL3 not detectable in TART, whereas INSL3 was highly expressed in LCTs. CONCLUSIONS: The similar expression profiles in TART and fetal adrenals as well as the presence of classical markers of adrenal steroidogenesis lend support to the hypothesis that TART develops from a displaced adrenal cell type. Malignant LCTs seem to have lost DLK1 expression and do not resemble immature LCs. The different expression pattern of DLK1, INSL3 and most adrenocortical markers adds to the elucidation of the histogenesis of testicular interstitial tumours and may facilitate histopathological diagnosis.

Genetics of primary macronodular adrenal hyperplasia.

ACTH-independent macronodular adrenal hyperplasia is a rare cause of Cushing's syndrome (CS), accounting for <2% of all endogenous CS cases; however it is more frequently identified incidentally with sub-clinical cortisol secretion. Recently, cortisol secretion has been shown to be regulated by ectopic corticotropin, which is in turn produced by clusters of steroidogenic cells of the hyperplastic adrenal nodules. Hence, the term 'ACTH-independent' is not entirely appropriate for this disorder. Accordingly, the disease is designated primary macronodular adrenal hyperplasia (PMAH) in this review article. The means by which cortisol production is regulated in PMAH despite the suppressed levels of ACTH of pituitary origin is exceedingly complex. Several molecular events have been proposed to explain the enhanced cortisol secretion, increased cell proliferation, and nodule formation in PMAH. Nonetheless, the precise sequence of events and the molecular mechanisms underlying this condition remain unclear. The purpose of this review is therefore to present new insights on the molecular and genetic profile of PMAH pathophysiology, and to discuss the implications for disease progression.

Replacement of short segments within transmembrane domains of MC2R disrupts retention signal.

The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1-24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).

A single nucleotide polymorphism in the corticotropin receptor gene is associated with a blunted cortisol response during pediatric critical illness.

OBJECTIVES: The cortisol response during critical illness varies widely among patients. Our objective was to examine single nucleotide polymorphisms in candidate genes regulating cortisol synthesis, metabolism, and activity to determine if genetic differences were associated with variability in the cortisol response among critically ill children. DESIGN: This was a prospective observational study employing tag single nucleotide polymorphism methodology to examine genetic contributions to the variability of the cortisol response in critical illness. Thirty-one candidate genes and 31 ancestry markers were examined. SETTING: Patients were enrolled from seven pediatric critical care units that constitute the Eunice Kennedy Shriver Collaborative Pediatric Critical Care Research Network. SUBJECTS: Critically ill children (n = 92), age 40 weeks gestation to 18 years old, were enrolled. INTERVENTIONS: Blood samples were obtained from all patients for serum cortisol measurements and DNA isolation. Demographic and illness severity data were collected. MEASUREMENTS AND MAIN RESULTS: Single nucleotide polymorphisms were tested for association with serum free cortisol concentrations in context of higher illness severity as quantified by Pediatric Risk of Mortality III score greater than 7. A single nucleotide polymorphism (rs1941088) in the MC2R gene was strongly associated (p = 0.0005) with a low free cortisol response to critical illness. Patients with the AA genotype were over seven times more likely to have a low free cortisol response to critical illness than those with a GG genotype. Patients with the GA genotype exhibited an intermediate free cortisol response to critical illness. CONCLUSIONS: The A allele at rs1941088 in the MC2R gene, which encodes the adrenocorticotropic hormone (corticotropin, ACTH) receptor, is associated with a low cortisol response in critically ill children. These data provide evidence for a genetic basis for a portion of the variability in cortisol production during critical illness. Independent replication of these findings will be important and could facilitate development of personalized treatment for patients with a low cortisol response to severe illness.

Development of potent selective competitive-antagonists of the melanocortin type 2 receptor.

Cushing's disease, a hypercortisolemic state induced by an ACTH overexpressing pituitary adenoma, causes increased morbidity and mortality. Selective antagonism of the melanocortin type 2 receptor (MC2R) may be a novel treatment modality. Five structurally related peptides with modified HFRW sites but intact putative MC2R binding sites were tested for antagonistic activity at MC1R, MC2R/MRAP, MC3R, MC4R and MC5R. Two of these peptides (GPS1573 and GPS1574) dose-dependently antagonized ACTH-stimulated MC2R activity (IC50s of 66±23 nM and 260±1 nM, respectively). GPS1573 and 1574 suppressed the Rmax but not EC50 of ACTH on MC2R, indicating non-competitive antagonism. These peptides did not antagonize α-MSH stimulation of MC1R and antagonized MC3, 4 and 5R at markedly lower potency. GP1573 and GPS1574 antagonize MC4R with IC50s of 950 nM and 3.7 µM, respectively. In conclusion, two peptide antagonists were developed with selectivity for MC2R, forming a platform for development of a medical treatment for Cushing's disease.

Increased expression of ACTH (MC2R) and androgen (AR) receptors in giant bilateral myelolipomas from patients with congenital adrenal hyperplasia.

BACKGROUND: Although chronic adrenocorticotropic hormone (ACTH) and androgen hyperstimulation are assumed to be involved in the pathogenesis of adrenal myelolipomas associated with poor-compliance patients with congenital adrenal hyperplasia (CAH), the expression of their receptors has not yet been demonstrated in these tumors so far. METHODS: We analyzed Melanocortin 2 receptor (MC2R), Androgen Receptor (AR), Leptin (LEP), and Steroidogenic factor 1 (SF1) expression using real-time qRT-PCR in two giant bilateral adrenal myelolipomas from two untreated simple virilizing CAH cases and in two sporadic adrenal myelolipomas. In addition, the X-chromosome inactivation pattern and CAG repeat numbers in AR exon 1 gene were evaluated in the 4 cases. RESULTS: The MC2R gene was overexpressed in myelolipomas from 3 out of 4 patients. AR overexpression was detected in 2 tumors: a giant bilateral myelolipoma in a CAH patient and a sporadic case. Simultaneous overexpression of AR and MC2R genes was found in two of the cases. Interestingly, the bilateral giant myelolipoma associated with CAH that had high androgen and ACTH levels but lacked MC2R and AR overexpression presented a significantly shorter AR allele compared with other tumors. In addition, X-chromosome inactivation pattern analysis showed a polyclonal origin in all tumors, suggesting a stimulatory effect as the trigger for tumor development. CONCLUSION: These findings are the first evidence for MC2R or AR overexpression in giant bilateral myelolipomas from poor-compliance CAH patients.

Expression and regulation of neuromedin B in pituitary corticotrophs of male melanocortin 2 receptor-deficient mice.

The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system that controls responses to stress, and has an important function in the regulation of various body processes. We previously created a mouse line deficient in the melanocortin 2 receptor (MC2R). MC2R-deficient mice (MC2R(-/-) mice) have high adrenocorticotropic hormone (ACTH) levels because of undetectable corticosterone levels. Increased neuromedin B (NMB) expression was recently reported in the pituitary gland of adrenalectomized mice, a model for acute adrenal insufficiency. To investigate gene expression in the pituitary gland under chronic adrenal deficiency, we examined the pituitary gland of MC2R(-/-) mice, a model of chronic adrenal insufficiency. To understand the molecular background of pituitary cells under chronic adrenal deficiency, we first performed DNA microarray analyses using the pituitary glands of the MC2R(-/-) mice. The DNA microarray analysis and real-time polymerase chain reaction showed that NMB expression was higher in the MC2R(-/-) than in the wild-type (WT) mice. We detected NMB expression in the MC2R(-/-) pituitary corticotrophs by immunohistochemistry using the specific antibodies for ACTH and NMB. In addition, the plasma NMB concentration was significantly higher in the MC2R(-/-) mice than in the WT mice. Subcutaneous implantation of a sustained-release corticosterone pellet decreased the expression of NMB mRNA as well as pituitary proopiomelanocortin mRNA. In isolated anterior pituitary cells, NMB mRNA expression was increased by the administration of corticotropin-releasing hormone (CRH) and was suppressed by dexamethasone treatment. In this study, we first demonstrate NMB expression in corticotrophs and its regulation by CRH and glucocorticoids. Furthermore, corticotrophs seemed to secrete NMB into the systemic circulation.

Effects of polycyclic musks HHCB and AHTN on steroidogenesis in H295R cells.

1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(γ)-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) are widely used in personal care products. Previous studies showed that HHCB and AHTN can be found in various environmental matrices and have potential endocrine disrupting effects. However, the effects on adrenocortical function of HHCB and AHTN are not fully understood. This study evaluated the influences of HHCB and AHTN on seven steroid hormones (progesterone, aldosterone, cortisol, 17α-OH-progesterone, androstenedione, 17ß-estradiol, and testosterone) and 10 genes involved in steroidogenic pathways (HMGR, StAR, CYP11A1, 3ßHSD2, CYP17, CYP21, CYP11B1, CYP11B2, 17ßHSD, and CYP19) using the H295R cell line in the absence and presence of 8-Br-cAMP. MC2R transcription on the cell membrane was also examined to further investigate the effects of HHCB and AHTN on adrenal steroidogenesis. The results demonstrated that HHCB and AHTN could inhibit progesterone and cortisol production mainly by the suppression of 3ßHSD2 and CYP21. Meanwhile, high concentrations of AHTN can affect the sensitivity of H295R cells to ACTH by disrupting MC2R transcription. Overall, the results indicate that high concentrations of HHCB and AHTN can affect steroidogenesis in vitro using the H295R cell line.