Molecular and pharmacological analysis of the melanocortin-2 receptor and its accessory proteins Mrap1 and Mrap2 in a Squalomorph shark, the Pacific spiny dogfish.

The hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is a conserved vertebrate neuroendocrine mechanism regulating the stress response. The penultimate step of the HPA/I axis is the exclusive activation of the melanocortin-2 receptor (Mc2r) by adrenocorticotropic hormone (ACTH), requiring an accessory protein, Mrap1 or Mrap2. Limited data for only three cartilaginous fishes support the hypothesis that Mc2r/Mrap1 function in bony vertebrates is a derived trait. Further, Mc2r/Mrap1 functional properties appear to contrast among cartilaginous fishes (i.e., the holocephalans and elasmobranchs). This study sought to determine whether functional properties of Mc2r/Mrap1 are conserved across elasmobranchs and in contrast to holocephalans. The deduced amino acid sequences of Pacific spiny dogfish (Squalus suckleyi; pd) pdMc2r, pdMrap1, and pdMrap2 were obtained from a de novo transcriptome of the interrenal gland and validated against the S. suckleyi genome. pdMc2r showed high primary sequence similarity with elasmobranch and holocephalan Mc2r except at extracellular domains 1 and 2, and transmembrane domain 5. pdMraps showed similarly high sequence similarity with holocephalan and other elasmobranch Mraps, with all cartilaginous fish Mrap1 orthologs lacking an activation motif. cAMP reporter gene assays demonstrated that pdMc2r requires an Mrap for activation, and can be activated by stingray (sr) ACTH(1-24), srACTH(1-13)NH2 (i.e., α-MSH), and γ-melanocyte-stimulating hormone at physiological concentrations. However, pdMc2r was three orders of magnitude more sensitive to srACTH(1-24) than srACTH(1-13)NH2. Further, pdMc2r was two orders of magnitude more sensitive to srACTH(1-24) when expressed with pdMrap1 than with pdMrap2. These data suggest that functional properties of pdMc2r/pdMrap1 reflect other elasmobranchs and contrast what is seen in holocephalans.

Functional characterization of melanocortin 2 receptor (Mc2r) from a lobe-finned fish (Protopterus annectens) and insights into the molecular evolution of melanocortin receptors.

Recent studies from our group on melanocortin 2 receptors (Mc2r) from basal families of actinopterygians have served to resolve that Mrap1 dependence and ACTH selectivity are features of even the most basal ray-finned fishes. However, there have been no studies on Mc2r function of the basal sarcopterygians, the lobe-finned fishes, represented by the extant members coelacanths and lungfishes. Here, we offer the first molecular and functional characterization of an Mc2r from a lobe-finned fish, the West African lungfish (Protopterus annectens). Plasmids containing cDNA constructs of lungfish (lf) Mc2r and Mrap1 were expressed in mammalian and zebrafish cell lines. Cells were then stimulated by human ACTH(1-24) and melanocyte stimulating hormone (α-MSH), as well as alanine-substituted analogs of hACTH(1-24) targeting residues within the H6F7R8W9 and K15K16R17R18P19 motifs. Activation of lfMc2r was assessed using a cAMP-responsive luciferase reporter gene assay. In these assays, lfMc2r required co-expression with lfMrap1, was selective for ACTH over α-MSH at physiological concentrations of the ligands, and was completely inhibited by multiple-alanine substitutions of the HFRW (A6-9) and KKRRP (A15-19) motifs. Single- and partial-alanine substitutions of the HFRW and KKRRP motifs varied in their impacts on receptor-ligand affinity from having no effect to completely inhibiting lfMc2r activation. This characterization of the Mc2r of a lobe-finned fish fulfills the last major extant vertebrate group for which Mc2r function had yet to be characterized. In doing so, we resolve that all basal bony vertebrate groups exhibit Mc2r function that substantially differs from that of the cartilaginous fishes, indicating that rapid and dramatic shift in Mc2r function occurred between the radiation of cartilaginous fishes and the emergence of bony fishes. We support this interpretation with a molecular clock analysis of the melanocortin receptors, which demonstrates the uniquely high rate of sequence divergence in Mc2r. Much remains to be understood regarding the molecular evolution of Mc2r during the early radiation of vertebrates that resulted in the derived functional characteristics of Mrap1 dependence and exclusive selectivity for ACTH.

Trends in the evolution of the elasmobranch melanocortin-2 receptor: Insights from structure/function studies on the activation of whale shark Mc2r.

To understand the mechanism for activation of the melanocortin-2 receptor (Mc2r) of the elasmobranch, Rhincodon typus (whale shark; ws), wsmc2r was co-expressed with wsmrap1 in CHO cells, and the transfected cells were stimulated with alanine-substituted analogs of ACTH(1-24) at the "message" motif (H6F7R8W9) and the "address" motif (K15K16R17R18P19). Complete alanine substitution of the H6F7R8W9 motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W9 > R8, and substitution at F7 and H6 had no effect on activation. The same analysis was done on a representative bony vertebrate Mc2r ortholog (Amia calva; bowfin; bf) and the order of position importance for activation was W9 > R8 = F7, (alanine substitution at H6 was negligible). Complete alanine substitution at the K15K16R17R18P19 motif resulted in distinct outcomes for wsMc2r and bfMc2r. For bfMc2r, this analog blocked activation-an outcome typical for bony vertebrate Mc2r orthologs. For wsMc2r, this analog resulted in a shift in sensitivity to stimulation of the analog as compared to ACTH(1-24) by two orders of magnitude, but the dose response curve did reach saturation. To evaluate whether the EC2 domain of wsMc2r plays a role in activation, a chimeric wsMc2r was made in which the EC2 domain was replaced with the EC2 domain from a melanocortin receptor that does not interact with Mrap1 (i.e., Xenopus tropicalis Mc1r). This substitution did not negatively impact the activation of the chimeric receptor. In addition, alanine substitution at a putative activation motif in the N-terminal of wsMrap1 did not affect the sensitivity of wsMc2r to stimulation by ACTH(1-24). Collectively, these observations suggest that wsMc2r may only have a HFRW binding site for melanocortin-related ligand which would explain how wsMc2r could be activated by either ACTH or MSH-sized ligands.

Hypothalamus-pituitary-interrenal (HPI) axis signaling in Atlantic sturgeon (Acipenser oxyrinchus) and sterlet (Acipenser ruthenus).

In vertebrates, the hypothalamic-pituitaryadrenal/interrenal (HPA/HPI) axis is a highly conserved endocrine axis that regulates glucocorticoid production via signaling by corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Once activated by ACTH, Gs protein-coupled melanocortin 2 receptors (Mc2r) present in corticosteroidogenic cells stimulate expression of steroidogenic acute regulatory protein (Star), which initiates steroid biosynthesis. In the present study, we examined the tissue distribution of genes involved in HPI axis signaling and steroidogenesis in the Atlantic sturgeon (Acipenser oxyrinchus) and provided the first functional characterization of Mc2r in sturgeon. Mc2r of A. oxyrinchus and the sterlet sturgeon (Acipenser ruthenus) are co-dependent on interaction with the melanocortin receptor accessory protein 1 (Mrap1) and highly selective for human (h) ACTH over other melanocortin ligands. A. oxyrinchus expresses key genes involved in HPI axis signaling in a tissue-specific manner that is indicative of the presence of a complete HPI axis in sturgeon. Importantly, we co-localized mc2r, mrap1, and star mRNA expression to the head kidney, indicating that this is possibly a site of ACTH-mediated corticosteroidogenesis in sturgeon. Our results are discussed in the context of other studies on the HPI axis of basal bony vertebrates, which, when taken together, demonstrate a need to better resolve the evolution of HPI axis signaling in vertebrates.

[Clinical characteristics and genetic analysis of two children with Familial glucocorticoid deficiency type 1 due to variants of MC2R gene].

OBJECTIVE: To improve the recognition of Familial glucocorticoid deficiency type 1 (FGD1) due to variants of melanocortin 2 receptor (MC2R) gene. METHODS: Two children with FGD1 diagnosed at the Henan Children's Hospital respectively in 2019 and 2021 were selected as the study subjects. Clinical data, treatment, follow-up and results of genetic testing were collected and retrospectively analyzed. RESULTS: Whole exome sequencing revealed that both children had harbored compound heterozygous variants of the MC2R gene, including c.433C>T (p.R145C) and c.710T>C (p.L237P) in child 1, and c.145delG (p.V49Cfs*35) and c.307G>A (p.D103N) in child 2, among which c.710T>C (p.L237P) and c.145delG (p.V49Cfs*35) were unreported previously. CONCLUSION: FGD1 is clinically rare, and genetic sequencing is crucial for the definite diagnosis. Discovery of the and novel variants has enriched the mutational spectrum of the FGD1 gene.

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.

Silver nanoparticles modify the hypothalamic-pituitary-interrenal axis and block cortisol response to an acute stress in zebrafish, Danio rerio.

The present study aimed at assessing the effects of exposure to silver nanoparticle (AgNP) and a subsequent acute stress on the expression of various genes involved in the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish, Danio rerio. The fish were exposed to 0 (Control), 0.1 (LC), 0.4 (MC), and 1.2 (HC) mg Ag/L (as AgNP) over a 2-week period, followed by an acute air exposure stress. The whole body cortisol and the expression of selected genes in the fish brain and kidney were analyzed, before and after the acute stress. The results showed that AgNP increased basal cortisol levels and the expression of corticotropin releasing factor, prohormone convertase 1, pro-opiomelanocortin, and melanocortin 2 receptor; however, it suppressed/inhibited whole body cortisol, brain corticotropin releasing factor responses, pro-opiomelanocortin, and the kidney melanocortin 2 receptor responses to the acute stress. AgNP down-regulated the expression of the steroidogenic acute regulatory protein, but it intensified the gene expression in response to the acute stress. Before the acute stress, LC treatment exhibited an up-regulation in Cytochrome P450-11A-1 expression, but MC and HC treatments induced down-regulation. After the acute stress, the AgNP-exposed fish exhibited decreased Cytochrome P450-11A-1 expressions, compared with the Control. Exposure to AgNP significantly increased Cytochrome P450-11B expression. However, after the acute stress, LC treatment exhibited an up-regulation, but MC and HC treatments exhibited down-regulation in the Cytochrome P450-11B gene expression. In conclusion, AgNP suppressed cortisol response to stress, which appears to be a consequence of alterations in the HPI axis at the transcriptomic levels.

New effects of caffeine on corticotropin-releasing hormone (CRH)-induced stress along the intrafollicular classical hypothalamic-pituitary-adrenal (HPA) axis (CRH-R1/2, IP3 -R, ACTH, MC-R2) and the neurogenic non-HPA axis (substance P, p75NTR and TrkA) in ex vivo human male androgenetic scalp hair follicles.

BACKGROUND: Human hair is highly responsive to stress, and human scalp hair follicles (HFs) contain a peripheral neuroendocrine equivalent of the systemic hypothalamic-pituitary-adrenal (HPA) stress axis. Androgenetic alopecia (AGA) is supposed to be aggravated by stress. We used corticotropin-releasing hormone (CRH), which triggers the HPA axis, to induce a stress response in human ex vivo male AGA HFs. Caffeine is known to reverse testosterone-mediated hair growth inhibition in the same hair organ culture model. OBJECTIVES: To investigate whether caffeine would antagonize CRH-mediated stress in these HFs. METHODS: HFs from balding vertex area scalp biopsies of men affected by AGA were incubated with CRH (10-7 mol L-1 ) with or without caffeine (0·001% or 0·005%). RESULTS: Compared to controls, CRH significantly enhanced the expression of catagen-inducing transforming growth factor-ß2 (TGF-ß2) (P < 0·001), CRH receptors 1 and 2 (CRH-R1/2) (P < 0·01), adrenocorticotropic hormone (ACTH) (P < 0·001) and melanocortin receptor 2 (MC-R2) (P < 0·001), and additional stress-associated parameters, substance P and p75 neurotrophin receptor (p75NTR ). CRH inhibited matrix keratinocyte proliferation and expression of anagen-promoting insulin-like growth factor-1 (IGF-1) and the pro-proliferative nerve growth factor receptor NGF-tyrosine kinase receptor A (TrkA). Caffeine significantly counteracted all described stress effects and additionally enhanced inositol trisphosphate receptor (IP3 -R), for the first time detected in human HFs. CONCLUSIONS: These findings provide the first evidence in ex vivo human AGA HFs that the stress mediator CRH induces not only a complex intrafollicular HPA response, but also a non-HPA-related stress response. Moreover, we show that these effects can be effectively antagonized by caffeine. Thus, these data strongly support the hypothesis that stress can impair human hair physiology and induce hair loss, and that caffeine may effectively counteract stress-induced hair damage and possibly prevent stress-induced hair loss.

Local secretion of stress hormones increases in alopecia areata lesions after treatment with UVA-1 phototherapy.

Alopecia areata (AA) is an autoimmune disease of the hair follicle. Keratinocytes of the hair follicle generate an immunosuppressive environment by the local secretion of hormones of the hypothalamic-pituitary-adrenal axis of the skin (skin HPA analog). Our objective was to measure the local production of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and α-melanocyte-stimulating hormone (α-MSH) in the scalp tissue of patients with AA before and after ultraviolet A1 (UVA-1) phototherapy to determine their role in the pathogenesis of AA and the effect of UVA-1 on the AA hormonal environment. This was a retrospective and descriptive study of skin samples from 22 patients with AA before and after UVA-1 treatment. We compared the changes in the local hormonal environment by measuring CRH, ACTH, type 2 melanocortin receptor (ACTH receptor) and α-MSH with immunohistochemical stains. The positivity of MSH was significantly higher (P = .037) in the post-treatment samples compared with the baseline value. ACTH was significantly higher in intensity (P = .032) in the post-treatment samples compared with the initial value. CRH was significantly higher in intensity (P = .013) in baseline samples compared with the final biopsies. The positivity of the ACTH receptor MC2R was not different between the two groups (P = .626). In AA, an interruption in the signalling of CRH could decrease the local concentration of ACTH and MSH, and consequently, the immunosuppressive effect of these hormones. This phenomenon is normalized in the skin treated with UVA-1. A defective signalling system in the cutaneous HPA axis may be involved in the pathogenesis of AA.

Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2.

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1-24) with an EC50 of 2.6 × 10-10 M ± 9.7 × 10-11. When the receptor was expressed alone, stimulation was only observed at [10-6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. In addition, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1-24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, and while co-expression with MRAP2 increases trafficking, it has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1-24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. Co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

Evaluating interactions between the melanocortin-5 receptor, MRAP1, and ACTH(1-24): A phylogenetic study.

The melanocortin-2 receptor (MC2R) and the melanocortin-5 receptor (MC5R) are found on the same chromosome in most vertebrate genomes, and for the species analyzed in this study, MC2R and MC5R are co-expressed in glucocorticoid-producing cells that also express the accessory protein MRAP1. Since MRAP1 affects the ligand sensitivity of MC2R orthologs, this study tested the hypothesis that co-expression of MC5R with MRAP1 would also affect the ligand sensitivity of MC5R. The hypothesis was confirmed for stingray, rainbow trout, and chicken, MC5R orthologs. However, elephant shark MC5R was not affected in the same way by co-expression of MRAP1. It appears that, for some MC5R orthologs (i.e., stingray, rainbow trout, and chicken), a docking site for the R/KKRRP motif of ACTH(1-24) may become exposed on the receptor following co-expression with MRAP1. However, for elephant shark MC5R co-expression with MRAP1 may not affect engagement ACTH(1-24). Hence during the radiation of the chordates, the interaction between MRAP1 and MC5R has diverged.

Elephant shark melanocortin receptors: Novel interactions with MRAP1 and implication for the HPI axis.

The presence of Mrap1 and Mrap2 orthologs in the genome of the elephant shark (es), a cartilaginous fish, presented an opportunity to evaluate the potential interactions between these accessory proteins and melanocortin receptors of a cartilaginous fish. RT-PCR analysis indicated that Mrap1 mRNA was present in interrenal, brain, and pituitary tissue with mRNA for Mc2R, Mc3R, Mc4R, and Mc5r. Co-expression of esMrap1 cDNA with esMc2r cDNA or esMc5r cDNA in CHO cells increased sensitivity to stimulation with ACTH(1-24) 10 fold and 100 fold, respectfully, but had no effect on sensitivity to stimulation with DesAc-αMSH [i.e., ACTH(1-13)NH2] for either receptor, and had no effect on the ligand sensitivity of either esMc3r or esMc4r. Fluorescence image analysis indicated co-localization of esMrap1/esMc2r, and esMrap1/esMc5r on the plasma membrane; however, cell surface ELISA analysis indicated that co-expression with esMrap1 had no effect, positive or negative, on the trafficking of either esMc2r or esMc5r to the plasma membrane. RT-PCR analysis also indicated that Mrap2 mRNA, as well as, mRNAs for Mc2r, Mc3r, Mc4r, and Mc5r could be detected in brain tissue, however no Mrap2 mRNA was detected in interrenal tissue. Co-expression of esMrap2 in CHO cells with, respectively, esMc2r, esMc4r, or esMc5r had no effect on ligand sensitivity. However, co-expression of esMrap2 with esMc3r did lower sensitivity to stimulation by DesAc-αMSH 10 fold. These observations are discussed in the context of the parallel evolution of melanocortin receptors and their accessory proteins, and the hypothalamus/pituitary/adrenal axis and the hypothalamus/pituitary/interrenal axis in bony vertebrates and cartilaginous fishes.

Identifying Common Features in the Activation of Melanocortin-2 Receptors: Studies on the Xenopus tropicalis Melanocortin-2 Receptor.

The interaction between the pituitary hormone, adrenocorticotropin (ACTH), and melanocortin-2 receptor (MC2R) orthologs involves the H6 F7 R8 W9 and R/K15 K16 R17 R18 motifs in ACTH making contact with corresponding contact sites on MC2R. Earlier studies have localized the common HFRW binding site of all melanocortin receptors to residues in TM2, TM3, and TM6 that are located close to the extracellular space. The current study has identified residues in Xenopus tropicalis (xt) MC2R in TM4 (I158, F161), in EC2 (M166), and in TM5 (V172) that also are involved in activation of xtMC2R, and may be in the R/KKRR contact site of xtMC2R. These results are compared to earlier studies on the corresponding domains of human MC2R and rainbow trout MC2R in an effort to identify common features in the activation of teleost and tetrapod MC2R orthologs following stimulation with ACTH.

Pathological cyclic strain promotes proliferation of vascular smooth muscle cells via the ACTH/ERK/STAT3 pathway.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is closely related to hyperplasia in hypertension. Our previous study suggested that adrenocorticotropic hormone (ACTH) is mechano-responsive and may regulate VSMC proliferation. However, the molecular mechanism of VSMC abnormal proliferation induced by conditions of high cyclic strain, especially the role of ACTH in this process, is unclear. Our results revealed that ACTH and its specific receptor melanocortin receptor type 2 (MC2R) were highly expressed in hypertensive rat models. Furthermore, it was demonstrated that the expression of ACTH and MC2R was up-regulated when exposed to high cyclic strain in vitro, accompanied by abnormal proliferation of VSMCs. Next, it was proved that ACTH-dependent cell proliferation was related to the phosphorylation of extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription 3 (STAT3). The study also found that ACTH could promote dimerization and glycosylation of melanocortin 2 receptor accessory protein (MRAP), which had a significant effect on MC2R membrane localization and signal activation. When VSMCs were treated with PD98059, a mitogen-activated protein kinase (MAP kinase) cascade antagonist, it was determined that phosphorylation of STAT3 at Ser727 was dependent on ERK phosphorylation. In summary, these data demonstrated that the abnormal proliferation of VSMCs induced by conditions of high cyclic strain is in part attributed to ACTH and its receptor MC2R. Identifying the mechanism of ACTH-dependent proliferation of VSMCs may help to provide new therapeutic targets for hypertension.

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.

Effect of a melanocortin type 2 receptor (MC2R) antagonist on the corticosterone response to hypoxia and ACTH stimulation in the neonatal rat.

The adrenal stress response in the neonatal rat shifts from ACTH-independent to ACTH-dependent between postnatal days 2 (PD2) and 8 (PD8). This may be due to an increase in an endogenous, bioactive, nonimmunoreactive ligand to the melanocortin type 2 receptor (MC2R). GPS1574 is a newly described MC2R antagonist that we have shown to be effective in vitro. Further experimentation with GPS1574 would allow better insight into this seemingly ACTH-independent steroidogenic response in neonates. We evaluated the acute corticosterone response to hypoxia or ACTH injection following pretreatment with GPS1574 (32 mg/kg) or vehicle for GPS1574 in PD2, PD8, and PD15 rat pups. Pretreatment with GPS1574 decreased baseline corticosterone in PD2 pups but increased baseline corticosterone in PD8 and PD15 pups. GPS1574 did not attenuate the corticosterone response to hypoxia in PD2 pups and augmented the corticosterone response in PD8 and PD15 pups. GPS1574 augmented the corticosterone response to ACTH in PD2 and PD15 pups but had no significant impact on the response in PD8 pups. Baseline adrenal Mrap and Star mRNA increased from PD2 to PD15, whereas Mrap2 mRNA expression was low and did not change with age. The data suggest that GPS1574 is not a pure MC2R antagonist, but rather acts as a biasing agonist/antagonist. Its ability to attenuate or augment the adrenal response may depend on the ambient plasma ACTH concentration and/or developmental changes in early transduction steroidogenic pathway genes.

The melanocortin-2 receptor of the rainbow trout: Identifying a role for critical positions in transmembrane domain 4, extracellular loop 2, and transmembrane domain 5 in the activation of rainbow trout MC2R.

The activation of either teleost or tetrapod melanocortin-2 receptor (MC2R) orthologs requires interaction between the HFRW motif and R/KKRRP motif in the primary sequence of ACTH, and two corresponding sites on the melanocortin 2 receptor. While the HFRW contact site on MC2R appears to involve residues in TM2, TM3, and TM6, several studies on human MC2R point to the EC2/TM5 region of MC2R as a possible location for the R/KKRRP contact site. In this study nineteen single-alanine mutants of rainbow trout (rt) MC2R were made beginning at V153 in TM4, at all positions in EC2 (extracellular loop 2), to F175 in TM5. For twelve of these alanine mutants (i.e., V153, G155, C162, D163, T165, V166, I167, H169, F170, H172, V173, L174), alanine substitution did not have a statistically significant effect on activation of the receptor. For four of these alanine mutations (i.e., V157, M158, F161, K168), while the negative shift in ligand sensitivity was statistically significant, the magnitude of the negative shift in activation was fivefold or less. However, for substitution at V159 in TM4 (negative shift in activation: 110 fold), F171 in TM5 (negative shift in activation: 48-fold), and F175 in TM5 (negative shift in activation: 100 fold), the effect on activation was both statistically significant and may be physiologically relevant. To support this conclusion, a triple alanine mutant of rtMC2R (V159/A, F171/A, F175/A), and this mutant receptor could not be activated by ACTH at concentrations as high as 10-6M. A Cell Surface ELISA analysis indicated that the trafficking of the triple alanine mutant rtMC2R to the plasma membrane was not impaired by the alanine substitutions. Collectively, these observations point to a critical role for TM4 and TM5 in the activation of the rainbow trout melanocortin-2 receptor.

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.

Effects of environmental noise exposure on DNA methylation in the brain and metabolic health.

Environmental noise exposure is associated with adverse effects on human health including hearing loss, heart disease, and changes in stress-related hormone levels. Alteration in DNA methylation in response to environmental exposures is a well-known phenomenon and it is implicated in many human diseases. Understanding how environmental noise exposures affect DNA methylation patterns may help to elucidate the link between noise and adverse effects on health. In this pilot study we examined the effects of environmental noise exposure on DNA methylation of genes related to brain function and investigated whether these changes are related with metabolic health. We exposed four groups of male Wistar rats to moderate intensity noise (70-75dB with 20-4000Hz) at night for three days as short-term exposure, and for three weeks as long-term exposure. Noise exposure was limited to 45dB during the daytime. Control groups were exposed to only 45dB, day and night. We measured DNA methylation in the Bdnf, Comt, Crhr1, Mc2r, and Snca genes in tissue from four brain regions of the rats (hippocampus, frontal lobe, medulla oblongata, and inferior colliculus). Further, we measured blood pressure and body weight after long-term noise exposure. We found that environmental noise exposure is associated with gene-specific DNA methylation changes in specific regions of the brain. Changes in DNA methylation are significantly associated with changes in body weight (between Bdnf DNA methylation and Δ body weight: r=0.59, p=0.018; and between LINE-1 ORF DNA methylation and Δ body weight: =-0.80, p=0.0004). We also observed that noise exposure decreased blood pressure (p=0.038 for SBP, p=0.017 for DBP and p 0. 017 for MAP) and decreased body weight (ß=-26g, p=0.008). In conclusion, environmental noise exposures can induce changes in DNA methylation in the brain, which may be associated with adverse effects upon metabolic health through modulation of response to stress-related hormones.

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.