α-Melanocyte-stimulating hormone inhibits angiogenesis through attenuation of VEGF/VEGFR2 signaling pathway.

BACKGROUND: Gene therapy of proopiomelanocortin, the precursor of α-melanocyte-stimulating hormone (α-MSH), suppresses the neovascularization in tumors. However, the roles of α-MSH in angiogenesis remain unclear. METHODS: The influence of α-MSH on angiogenesis was evaluated by ex vivo rat aorta and in vivo, including transgenic zebrafish and chicken chorioallantoic membrane (CAM) assays. The effect of α-MSH on proliferation, matrix metalloproteinase (MMP) secretion, migration and tube formation was examined using human umbilical vein endothelial cells (HUVECs). The expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) was investigated by quantitative RT-PCR, immunoblot and immunofluorescent analysis. Antibodies' neutralization was employed to dissect the receptor(s) transmitting α-MSH signaling. RESULTS: Application of α-MSH potently suppressed the microvessels sprouting in organotypic aortic rings. Besides, α-MSH perturbed the vessels development in zebrafish and chicken embryos. α-MSH (0.01-10nM) inhibited the MMP-2 secretion, migration and tube formation of HUVECs without affecting proliferation. Mechanistic studies unveiled α-MSH decreased the VEGF expression and release in HUVECs. Besides, α-MSH downregulated the VEGFR2 expression at transcriptional and translational levels. Importantly, α-MSH attenuated the Akt phosphorylation, but enhanced the expression of PTEN, endogenous antagonist of PI3K/Akt signaling. Expression analysis and antibody neutralization revealed that MC1-R and MC2-R participated in α-MSH-induced blockage of migration and VEGF/VEGFR2/Akt signaling. However, VEGF supply failed to reverse the anti-angiogenic function of α-MSH. CONCLUSIONS: α-MSH inhibits the physiological angiogenesis by attenuating VEGF/VEGFR2/Akt signaling in endothelial cells. GENERAL SIGNIFICANCE: α-MSH is a potent angiogenesis inhibitor targeting at endothelial VEGF/VEGFR2 signaling, which may have potential for therapeutic application.

Further evidence on acetylation-induced inhibition of the pigment-dispersing activity of α-melanocyte-stimulating hormone.

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.

Observations on the ligand selectivity of the melanocortin 2 receptor.

The melanocortin 2 receptor (MC2R) is unique in terms of ligand selectivity and in vitro expression in mammalian cell lines as compared to the other four mammalian MCRs. It is well established that ACTH is the only melanocortin ligand that can activate the ACTH receptor (i.e., melanocortin 2 receptor). Recent studies have provided new insights into the presence of a common binding site for the HFRW motif common to all melanocortin ligands. However, the activation of the melanocortin 2 receptor requires an additional amino acid motif that is only found in the sequence of ACTH. This mini-review will focus on these two topics and provide a phylogenetic perspective on the evolution of MC2R ligand selectivity.

Localisation of the melanocortin-2-receptor and its accessory proteins in the developing and adult adrenal gland.

The melanocortin-2-receptor (MC2R)/MC2R accessory protein (MRAP) complex is critical to the production of glucocorticoids from the adrenal cortex. Inactivating mutations in either MC2R or MRAP result in the clinical condition familial glucocorticoid deficiency. The localisation of MC2R together with MRAP within the adrenal gland has not previously been reported. Furthermore, MRAP2, a paralogue of MRAP, has been shown in vitro to have a similar function to MRAP, facilitating MC2R trafficking and responsiveness to ACTH. Despite similar MC2R accessory functions, in vivo, patients with inactivating mutations of MRAP fail to be rescued by a functioning MRAP2 gene, suggesting differences in adrenal expression, localisation and/or function between the two MRAPs. In this study on the rat adrenal gland, we demonstrate that while MRAP and MC2R are highly expressed in the zona fasciculata, MRAP2 is expressed throughout the adrenal cortex in low quantities. In the developing adrenal gland, both MRAP and MRAP2 are equally well expressed. The MC2R/MRAP2 complex requires much higher concentrations of ACTH to activate compared with the MC2R/MRAP complex. Interestingly, expression of MC2R and MRAP in the undifferentiated zone would support the notion that ACTH may play an important role in adrenal cell differentiation and maintenance.

Age-related changes in pro-opiomelanocortin (POMC) and related receptors in human epidermis.

SYNOPSIS: Much effort has been placed in cosmetic research for better understanding of the effects of ageing on skin's appearance, structure, mechanical properties and function. It is now of common knowledge that UV radiations induce pre-mature skin ageing notably in the epidermis where UV radiations induce keratinocyte differentiation. As UV radiations have also been shown to regulate the pro-opiomelanocortin (POMC) peptide family in the skin and because no study has been conducted so far to investigate the age-related changes in POMC and related receptors, we analysed POMC, MC-1R, MC-2R and MOR-1 at mRNA level and MC-1R, MC-2R and MOR-1 at protein level too in primary cultures of normal human keratinocytes obtained from female donors aged from 17 to 75 years old. Regarding the gene expressions, we observed that MC-1R, MC-2R and MOR-1 suffered a dramatic decrease after 50 years of age, whereas POMC increased five-fold. Western blot analysis confirmed these results except for MOR-1 whose expression appeared to decrease at older age, around 70 years old. Immunostainings specific to MC-1R, MC-2R and MOR-1 performed on full-thickness skin biopsies also revealed an intense staining in the basal and spinous layers of a 30-year-old donor, whereas no reactivity could be observed in a 60-year-old one. We conclude that POMC and POMC-related receptors suffer a dramatically disturbed balance with ageing and that this may be implicated in the general process of skin ageing.

ACTH receptor blockade: a novel approach to treat congenital adrenal hyperplasia, or Cushing's disease.

It is hypothesized that blocking the adrenocorticotrophic hormone (ACTH) receptor, using either a blocking antibody or a drug will result in a medical cortical-adrenalectomy, with relative sparing of mineralocorticoid hormone production. This would be similar to the clinical findings in familial glucocorticoid deficiency type 1, an autosomal recessive condition due to inactivating mutations of the adrenal receptor for ACTH, also known as the melanocortin 2 receptor (MC2R). It is further hypothesized that MC2R blockade should allow using lower glucocorticoid doses to treat congenital adrenal hyperplasia (CAH) due to enzyme deficiency of either 21-hydroxylase (CYP21B) or 11-hydroxylase (CYP11B1), thus reaching a better final adult height than with current therapeutic strategies. Blocking the ACTH receptor can also be employed to medically treat Cushing's due to excess pituitary or ectopic ACTH production.

Role of asparagine-linked glycosylation in cell surface expression and function of the human adrenocorticotropin receptor (melanocortin 2 receptor) in 293/FRT cells.

Asparagine-linked glycosylation (N-glycosylation) of G protein-coupled receptors may be necessary for functions ranging from agonist binding, folding, maturation, stability, and internalization. Human melanocortin 2 receptor (MC2R) possesses putative N-glycosylation sites in its N-terminal extracellular domain; however, to date, the role of MC2R N-glycosylation has yet to be investigated. The objective of the present study is to examine whether N-glycosylation is essential or not for cell surface expression and cAMP production in native and MC2R accessory protein (MRAP alpha, -beta, or -dCT)-expressing cells using 293/FRT transfected with Myc-MC2R. Western blot analyses performed with or without endoglycosidase H, peptide:N-glycosidase F or tunicamycin treatments and site-directed mutagenesis revealed that MC2R was glycosylated in the N-terminal domain at its two putative N-glycosylation sites (Asn(12)-Asn(13)-Thr(14) and Asn(17)-Asn(18)-Ser(19)). In the absence of human MRAP coexpression, N-glycosylation of at least one of the two sites was necessary for MC2R cell surface expression. However, when MRAP was present, cell surface expression of MC2R mutants was either rescued entirely with the N17-18Q (QQNN) and N12-13Q (NNQQ) mutants or partially with the unglycosylated N12-13, 17-18Q (QQQQ) mutant. Functional and expression analyses revealed a discrepancy between wild-type (WT) and QQQQ cell surface receptor levels and maximal cAMP production with a 4-fold increase in EC(50) values. Taken together, these results indicate that the absence of MC2R N-glycosylation abrogates to a large extent MC2R cell surface expression in the absence of MRAPs, whereas when MC2R is N-glycosylated, it can be expressed at the plasma membrane without MRAP assistance.

Modeling the evolution of the MC2R and MC5R genes: studies on the cartilaginous fish, Heterondotus francisci.

Comparative studies support the hypothesis that the proliferation of melanocortin receptor genes (MCRs) in gnathostomes corresponds to the 2R hypothesis for the radiation of gene families in Phylum Chordata. This mini-review will initially focus on the distribution of MCRs in cartilaginous fish and the relationship between the shark MC5R gene and the proposed ancestral MC5R/2R gene. This section will be followed by the results of recent studies on the features of the ligand binding site common to all melanocortin receptors. These data will provide the background for a set of hypotheses to explain the unique ligand selectivity of the MC2 receptor in teleosts and tetrapods.

A novel adrenocorticotropin receptor mutation alters its structure and function, causing familial glucocorticoid deficiency.

CONTEXT: Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by unresponsiveness to ACTH. In this study, two mutations of the ACTH receptor (MC2R) gene are reported in this FGD clinical case. OBJECTIVE: The objective of the study was to characterize a novel MC2R gene mutation in a compound heterozygous patient with FGD phenotype. DESIGN: This was a clinical case description, biochemical, molecular, and bioinformatics analysis to describe a novel MC2R gene mutation. PATIENTS: The subject of the study was a male diagnosed with primary adrenal insufficiency. The family history showed nonconsanguineous healthy parents, three healthy siblings, and one brother affected with FGD. MAIN OUTCOME MEASURES: The mutant MC2R-Ala126Ser showed significantly lower activity when it was stimulated with ACTH-(1-24) than did cells transfected with wild-type MC2R. RESULTS: The molecular studies demonstrated the presence of an adenine heterozygous insertion (InsA1347) in the MC2R gene (G217fs) in the patient. This insertion was due to a frame shift mutation in one allele and a premature stop codon codifying an aberrant receptor of 247 residues (27.2 kDa). We also found a novel heterozygous mutation alanine 126 by serine. Molecular dynamic simulations showed that serine 126 side chain fluctuates forming a noncanonical intrahelical hydrogen bond in the transmembrane helix 3 of the mutated receptor. This produces a structural rearrangement of the MC2R internal cavities that may affect the ligand recognition and signal transduction throughout the G protein. CONCLUSIONS: We propose a molecular explanation for the reduced activity exhibited by the MC2R alanine 126 by serine mutant.

Adrenocorticotropin resistance syndromes.

Familial glucocorticoid deficiency (FGD) and triple A syndrome belong to a rare group of autosomal recessive disorders characterized by adrenocorticotropin (ACTH) insensitivity. Unlike triple A syndrome which presents a range of clinical features, FGD is solely characterized by glucocorticoid deficiency. ACTH regulates steroid biosynthesis in the adrenal cortex by exerting its effects via the ACTH receptor (melanocortin- 2 receptor, MC2R). In FGD, mutations in the MC2R account for only approximately 25% of cases (FGD type 1). The inability to express a functional MC2R in non-adrenal cell lines had implied the presence of an adrenal specific accessory factor(s), essential for MC2R expression. More recently, this factor was identified as melanocortin receptor accessory protein (MRAP). Mutations in MRAP account for 20% of cases (FGD type 2). Like the receptor activity-modifying proteins (RAMPs) and receptor transporter proteins (RTPs), which are well-characterized accessory proteins for G-protein-coupled receptors (GPCRs), MRAP is a small single transmembrane domain protein. MRAP is essential for the functional expression of the MC2R. About 55% of FGD cases have no identifiable gene defect, implying the involvement of additional genes. This chapter briefly describes the clinical and biochemical features of ACTH resistance syndromes. However, we will focus on the recent progress made towards understanding the molecular defect underlying these conditions, in particular the interaction of MC2R and MRAP.

The melanocortin 2 receptor accessory protein exists as a homodimer and is essential for the function of the melanocortin 2 receptor in the mouse y1 cell line.

The ACTH receptor [melanocortin 2 receptor (MC2R)] gene produces a functional receptor only when transfected into cells of adrenocortical origin, implying that it may require an adrenal-specific accessory factor. Recently we showed that the MC2R accessory protein (MRAP) is essential for the cell surface expression of the MC2R in such models. Using RNA interference (RNAi) technology, we have further explored the action of MRAP in the functioning of the MC2R in Y1 mouse adrenocortical cells that endogenously express MRAP and MC2R. We created stable cell lines expressing mouse MRAP short hairpin RNA (shRNAs) by transfecting cells with an expression vector containing the MRAP small interfering RNA sequence. The knockdown of MRAP resulted in a reduction in MC2R signaling. The overexpression of a mouse MRAP-Flag construct did not restore the expression of MRAP due to its degradation by the mouse shRNAs. The introduction of human MRAP that is resistant to silencing by mouse MRAP shRNAs resulted in the rescue of the MC2R signaling. MRAP migrates on SDS-PAGE with markedly lower mobility than predicted for a 14.1-kDa protein. Coimmunoprecipitation and mass spectroscopy suggests that MRAP exists as a homodimer that is resistant to dissociation by sodium dodecyl sulfate and reducing agents.

Familial glucocorticoid deficiency: advances in the molecular understanding of ACTH action.

Familial glucocorticoid deficiency (FGD), otherwise known as hereditary unresponsiveness to ACTH, is a rare autosomal recessive disease characterized by glucocorticoid deficiency in the absence of mineralocorticoid deficiency. Mutations of the ACTH receptor, also known as the melanocortin-2 receptor (MC2R), account for approximately 25% of FGD cases. More recently a second gene, MRAP (melanocortin-2 receptor accessory protein), was identified and found to account for a further 15-20%. MRAP encodes a small single transmembrane domain protein, which is essential in the trafficking of the MC2R to the cell surface. In this review, we will firstly summarize the clinical presentation and genetic aetiology of this condition. Secondly, we will discuss how the discovery of MRAP has enhanced our understanding of the mechanisms of ACTH/MC2R action. Finally, we will explore future developments in this field.

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis.

ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

Structural insights into the role of the ACTH receptor cysteine residues on receptor function.

The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH(2) terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.

Mechanisms of disease: the adrenocorticotropin receptor and disease.

The action of the peptide hormone adrenocorticotropin (ACTH) to stimulate glucocorticoid production by the adrenal gland is an essential physiologic process, yet is dependent on a single unique genetic component--the ACTH receptor or melanocortin 2 receptor. Genetic defects that cause abnormalities in this receptor or in a protein required for its expression at the cell surface result in a potentially fatal disease (familial glucocorticoid deficiency). Overexpression of this receptor or inability to desensitize it is found in adrenal adenomas or hyperplasia associated with glucocorticoid overproduction (Cushing syndrome). These disorders are uncommon, but there are considerable data to show that the hypothalamo-pituitary-adrenal axis is overactive, or in some circumstances underactive, in more common situations including depressive illness and septic shock. The origin of these latter disturbances is undoubtedly complex and multifactorial, but there is good evidence that a component of this phenomenon is an altered responsiveness of the ACTH receptor to ACTH. Understanding the basis of ACTH responsiveness might, therefore, contribute to the understanding of disorders such as these and perhaps enable the hypothalamo-pituitary-adrenal axis to be manipulated beneficially in these circumstances.

Adrenocorticotropic hormone-mediated signaling cascades coordinate a cyclic pattern of steroidogenic factor 1-dependent transcriptional activation.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that has emerged as a critical mediator of endocrine function at multiple levels of the hypothalamic-pituitary-steroidogenic axis. Within the adrenal cortex, ACTH-dependent transcriptional responses, including transcriptional activation of several key steroidogenic enzymes within the steroid biosynthetic pathway, are largely dependent upon SF-1 action. The absence of a bona fide endogenous eukaryotic ligand for SF-1 suggests that signaling pathway activation downstream of the melanocortin 2 receptor (Mc2r) modulates this transcriptional response. We have used the chromatin immunoprecipitation assay to examine the temporal formation of ACTH-dependent transcription complexes on the Mc2r gene promoter. In parallel, ACTH-dependent signaling events were examined in an attempt to correlate transcriptional events with the upstream activation of signaling pathways. Our results demonstrate that ACTH-dependent signaling cascades modulate the temporal dynamics of SF-1-dependent complex assembly on the Mc2r promoter. Strikingly, the pattern of SF-1 recruitment and the subsequent attainment of active rounds of transcription support a kinetic model of SF-1 transcriptional activation, a model originally established in the context of ligand-dependent transcription by several classical nuclear hormone receptors. An assessment of the major ACTH-dependent signaling pathways highlights pivotal roles for the MAPK as well as the cAMP-dependent protein kinase A pathway in the entrainment of SF-1-mediated transcriptional events. In addition, the current study demonstrates that specific enzymatic activities are capable of regulating distinct facets of a highly ordered transcriptional response.

Evidence for direct actions of melanocortin peptides on bone metabolism.

Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.

A peroxisome proliferator-response element in the murine mc2-r promoter regulates its transcriptional activation during differentiation of 3T3-L1 adipocytes.

Adrenocorticotropic hormone can stimulate lipolysis and suppress leptin expression in murine adipocytes. These effects are mediated via the melanocortin 2 receptor (MC2-R), which is expressed when 3T3-L1 cells are induced to undergo adipogenesis. In this study, we have characterized the mc2-r promoter in the murine adipocyte, one of the few extra-adrenal sites of expression and a cell type that lacks steroidogenic factor 1 (SF-1), a transcription factor that is required for mc2-r expression in adrenal cells. Transcriptional regulation of the mc2-r in the absence of SF-1 was investigated by 5' deletion analysis of the murine mc2-r promoter in both undifferentiated and differentiated 3T3-L1 cells. The results revealed the presence of a 59-base pair regulatory region within the promoter containing an adipocyte-specific enhancer. The ability of this region to confer enhanced activity in the adipocyte was mapped to a peroxisome proliferator-response element (PPRE)-like sequence that bound to peroxisome proliferator-activated receptor gamma (PPARgamma) and its heterodimeric partner retinoid X receptor alpha (RXRalpha) in adipocyte nuclear extracts. Co-transfection of PPARgamma2/RXRalpha with the pMC2-R(-112/+105)GL3 reporter resulted in transcriptional activation in preadipocytes, and this response required an intact PPRE. Mutation of the PPRE to prevent PPARgamma/RXRalpha binding resulted in a complete abrogation of the pMC2-R(-112/+105)GL3 reporter activity in day 3 differentiated 3T3-L1 cells, demonstrating a key role played by this site in regulating MC2-R expression in the murine adipocyte. These data highlight a novel mechanism for mc2-r transcription, which may have significance in both adrenal and extra-adrenal sites of expression.