Targeting Melanocortins to Control Inflammation

[Semin]

Targeting Melanocortin Receptors as a Novel Strategy to Control Inflammation
2004

Abstract
Adrenocorticotropic hormone and ?-, ?-, and ?-melanocyte-stimulating hormones, collectively called melanocortin peptides, exert multiple effects upon the host. These effects range from modulation of fever and inflammation to control of food intake, autonomic functions, and exocrine secretions. Recognition and cloning of five melanocortin receptors (MCRs) has greatly improved understanding of peptide-target cell interactions. Preclinical investigations indicate that activation of certain MCR subtypes, primarily MC1R and MC3R, could be a novel strategy to control inflammatory disorders. As a consequence of reduced translocation of the nuclear factor ?B to the nucleus, MCR activation causes a collective reduction of the major molecules involved in the inflammatory process. Therefore, anti-inflammatory influences are broad and are not restricted to a specific mediator. Short half-life and lack of selectivity could be an obstacle to the use of the natural melanocortins. However, design and synthesis of new MCR ligands with selective chemical properties are in progress.

Introduction
Adrenocorticotropic hormone (ACTH) and ?-, ?-, and ?-melanocyte-stimulating (?-, ?-, ?-MSH) hormones derive from post-translational processing of the precursor molecule proopiomelanocortin (POMC). These POMC products are collectively called melanocortin peptides or melanocortins. Although adrenal stimulatory effects of ACTH and pigmentary influences of MSH have been known for years, the discovery that melanocortin peptides have multiple effects on the host is much more recent. These effects are disparate and range from modulation of fever and inflammation to control of food intake, autonomic functions, and exocrine secretions. Furthermore, recent research indicates that certain melanocortin peptides have antimicrobial effects.

Recognition and cloning of melanocortin receptors (MCRs) has greatly improved understanding of peptide-target cell interactions. Synthetic melanocortins with selective affinities for individual MCR may soon form the basis for new classes of therapeutic molecules. In this article we summarize current physiological and pharmacological knowledge on melanocortins and their receptors and discuss possible therapeutic targets with a focus on treatment of inflammatory disorders. Treatment of obesity and sexual dysfunction are other significant therapeutic targets for receptor-specific melanocortins; for these topics we refer the readers to recent reviews. A detailed description of effects of melanocortins in skin physiology and melanocyte function and influences on behavior, learning, and memory are likewise beyond the scope of this review.

A. Proopiomelanocortin Gene
In 1977, a common precursor for ACTH/?-MSH and ?-lipotropin (?-LPH)/?-MSH/?-endorphin was found in mouse AtT-20 pituitary tumor cells. In 1979, analysis of the nucleotide sequence of cloned cDNA of bovine POMC showed a hitherto unknown MSH-like peptide sequence, called ?-MSH, as well as other N-terminal peptides. Gene sequences for human and rat POMC were described shortly thereafter. POMC sequence was subsequently determined in several species of mammals, amphibians, and teleosts; all the sequences revealed the same structural organization. In humans, there is a single POMC gene per haploid nucleus located on chromosome 2p23. The pituitary of lamprey, the most ancient of vertebrates, contains recognizable POMC sequences with structural similarity to those of teleosts and higher vertebrates, suggesting that POMC was present in common ancestors of lampreys and gnathostomes some 700 million or more years ago. In humans, the POMC gene is unusual in that it possesses three promoter regions that control transcription]
B. Proopiomelanocortin Gene Expression
In addition to the pituitary, where it was originally found, POMC expression and peptide processing occur normally in the nervous system and in widespread peripheral tissues. In the brain, POMC cell bodies are found in the hypothalamic arcuate nucleus and nucleus of solitary tract in the caudal brainstem. POMC mRNA is also detectable in the spinal cord and dorsal root ganglion. Within the hypothalamus, the integrating center for energy balance, POMC neurons have extensive interactions with other pathways. Melanocortinergic terminals are found in various hypothalamic regions such as paraventricular, dorsomedial hypothalamic nucleus, arcuate nucleus, and lateral hypothalamic regions. The POMC neurons in the arcuate nucleus express leptin receptors, through which leptin regulates POMC expression. The arcuate nucleus POMC neurons also express neuropeptide Y1 and Y5 receptors, receive neuropeptide Y innervations, and interact with neuropeptide Y/agouti gene-related protein (AgRP) neurons locally. POMC neurons project broadly to many brain regions, including those hypothalamic and brainstem nuclei important for regulating energy homeostasis. Furthermore, POMC neurons send projections to sympathetic preganglionic neurons in the thoracic spinal cord.

Although ectopic POMC syndrome associated with malignancies has been known for decades knowledge that POMC is expressed also in normal tissues is more recent. Especially important to inflammation, POMC mRNA also occurs in lymphocytes, monocytes, keratinocytes, and melanocytes, and it is clear that POMC peptides have regulatory functions in these cells. There is evidence that leukemia inhibitory factor stimulates POMC expression via phosphorylation of signal transducers and activators of transcription (STATs) STAT1 and STAT3 proteins. Therefore, activation of the STAT signaling pathway by cytokines, interferons, or hormones can increase POMC expression and melanocortin peptide production at sites of infection or inflammation. It appears that there are multiple forms of POMC transcripts. Pituitary POMC mRNA encodes a secreted protein, whereas certain brain and peripheral cells express a truncated POMC mRNA without coding for a signal sequence.

C. Post-Translational Processing of Proopiomelanocortin
Biologically active proteins and peptides are often generated by intracellular proteolysis of inactive precursors. This evolutionarily ancient mechanism depends on the production of specific secretory enzymes and the tight regulation of their activities. Such processing enzymes usually cleave proproteins at selected sites composed of single or paired basic amino acids. The latter are found in precursors of most of neural and peptide hormones, proteolytic enzymes, growth factors receptors, and signaling molecules. Thus, generation of biologically active peptides and proteins depends on two main components]
The secretory enzymes responsible for intracellular cleavage of POMC have been characterized. They belong to a family of serine proteinases of the subtilisin/kexin-type. There are seven known mammalian precursor (or proprotein) convertases (PCs) cleaving at single and/or pairs of basic residues]
Analysis of tissue expression and cellular localization of the convertases showed that only PC1 and PC2 are found in dense secretory granules. These enzymes have a key role in the processing of neuropeptide and endocrine precursors whose products are stored in granules. All the other enzymes concentrate and act at the level of the trans-Golgi network, en route to the cell surface, or at the level of plasma membrane. Thus, the latter group of enzymes appears to be primarily responsible for processing precursors whose products reach the cell surface or are secreted constitutively.

When PC1 and PC2 were discovered, POMC was soon identified as a potential substrate. This idea was supported by the observation of enhanced expression of such enzymes in pituitary POMC-producing cells. Data showed that PC1 generated ACTH and ?-LPH, whereas PC2 was required for production of ?-MSH and ?-endorphin. This was the first evidence that tissue-specific processing of POMC could be explained by the relative expression of its convertases. Thus, in the corticotrophs where PC1 predominates, ACTH and ?-LPH are the final POMC-processing products. In contrast, expression of PC2 in the pituitary pars intermedia accounts for production of ?-MSH and ?-endorphin. PACE4 and furin can also generate ACTH and ?-LPH both in the pituitary and in extrapituitary sites. Indeed, it is clear that POMC, PC1, and PC2, as well as other convertases, are expressed in extrapituitary tissues including the immune system and the skin.

D. Melanocortin Peptides
Amino acid sequences of human melanocortin peptides ?- and ?-MSH and ACTH were purified and sequenced in the 1950s. ?-MSH was found to share the sequence of ACTH (1-13), although ?-MSH is acetylated at the N terminus and C-terminally amidated. The structure of the ?-MSH peptides of different vertebrates is more variable than that of ?-MSH. ?-MSH was originally isolated from human pituitaries as a side fraction of somatotropin preparation and thereafter found to correspond to the 37-58 region of human ?-LPH. Subsequently, the peptide was considered to be an artifact of procedure and did not exist in humans. However, more recently, naturally occurring ?-MSH-octadecapeptide has been identified in human hypothalamus. The peptide corresponds with the ?-LPH (41-58) sequence. Subsequently, POMC was found to contain yet another MSH peptide sequence, ?-MSH. All the melanocortin peptides share an “invariant” sequence of four amino acids, His-Phe-Arg-Trp, which are the residues 6-9 in ACTH and ?-MSH.

Melanocortin Receptors and Their Endogenous Antagonists
The five MCRs cloned so far belong to the class A of guanine nucleotide-binding protein (G protein)-coupled, seven transmembrane receptors. They are the product of small genes, many of which are polymorphic. The MCRs show high sequence homologies, ranging from 60% identity between MC4R and MC5R, to 38% identity between MC2R and MC4R. MCRs are the smallest G protein-coupled receptors known, with short amino- and carboxyl-terminal ends and a very small second extracellular loop. All are functionally coupled to adenylyl cyclase and mediate their effects primarily by activating a cAMP-dependent signaling pathway.

A. MC1 Receptor
MC1R was the first member of the MCR gene family to be cloned. The cloned cDNA encoded a 317-amino acid protein with the transmembrane topography characteristic of receptors that couple to heterotrimeric G proteins. The relative affinity of the human MC1R for the natural melanocortins is ?-MSH ? ACTH > ?-MSH >> ?-MSH. These differences in affinity reproduce the relative potency of the melanocortin peptides in stimulation of melanogenesis and explain the lack of melanogenic activity of ?-MSH.

?-MSH/MC1R interactions contribute to regulation of skin physiology and melanogenesis. Binding of ?-MSH to its MC1R in melanocytes starts a signal cascade that activates adenylyl cyclase, increases intracellular cAMP, and induces activity of tyrosinase, the rate-limiting enzyme in the eumelanin synthetic pathway. MC1R mRNA in the skin is up-regulated by its own melanocortin ligands and by endothelin-1. Furthermore, MC1R expression appears to be regulated by the microphthalmia-associated transcription factor (MITF). This transcription factor belongs to the family of bHLH-LZ type transcription factors and promotes transcription of genes for melanogenesis-related enzymes such as tyrosinase. Although promoter deletion and transactivation studies failed to demonstrate direct MC1R activation by MITF through this site, in coexpression studies, induction of MC1R promoter activity increased by 5-fold in the presence of MITF. In addition to effects in melanocytes, there is evidence that MITF enhances expression of MC1R in cultured murine mast cells.

It is clear that MC1R functions extend well beyond regulation of melanogenesis. MC1R expression occurs in macrophage/monocytic cells, lymphocytes with antigen-presenting and cytotoxic functions, neutrophils, endothelial cells, astrocytes, and fibroblasts. Peripheral blood-derived dendritic cells were likewise found to express MC1R. Although this receptor subtype occurs mainly in peripheral tissues, in situ hybridization and immunohistochemistry techniques demonstrated its expression also in scattered neurons of periaqueductal gray substance in rat and human brains. Transactivation of MC1R in inflammatory cells causes marked reduction of activation and translocation to the nucleus of the transcription factor NF-?B. Consequently, there are marked anti-inflammatory effects exerted through inhibition of NF-?B-mediated transcription.

Flow cytometry studies showed that MC1R is expressed by in vitro-activated monocytes/macrophages and by the THP-1 monocytic cell line, at ratios of approximately one third to one fifth that of melanoma cells. However, although MC1R in immunocytes and endothelial cells is activated by picomolar concentration of ?-MSH, MC1R activation in melanocytes requires nanomolar concentrations of the peptide. Therefore, although receptor density in inflammatory cells is less than that in melanocytes, it appears that receptor affinity is much greater.

B. MC2 Receptor
The melanocortin-2 receptor (MC2R), also known as ACTH receptor, is selectively activated by adrenocorticotropic hormone. The ACTH receptor/MC2R gene was originally isolated by homology screening of human cDNA and genomic DNA libraries. The ACTH receptor gene encodes a 297-amino acid G protein-coupled receptor and shows the characteristic seven transmembrane-spanning domains that form the ligand binding site.

The physiological influences of ACTH on production and release of steroids by the adrenal cortex, their circadian variation, and stress-related fluctuations are mediated by MC2R. Binding of ACTH to its receptor stimulates adenylyl cyclase and induces increases in cell cAMP; this leads to activation of PKA, which promotes expression of steroidogenic enzymes.

In situ hybridization studies revealed dense expression of MC2R in the zona glomerulosa and zona fasciculata of the adrenal cortex, the sites of mineralocorticoid and glucocorticoid production. The zona reticularis showed less mRNA labeling. In the adrenal medulla, only scattered cells with unknown functions stain for MC2R mRNA. MC2R expression in adrenal cells is up-regulated by its ligand ACTH.

In addition to the adrenal glands, the MC2R mRNA has been found in murine adipocytes, where it is believed to mediate stress-induced lipolysis in response to ACTH. However, ACTH does not appear to regulate adipocyte function in humans and other primates, as human adipocytes lack expression of MC2R. Furthermore, recent research indicated failure to export the ACTH receptor from the endoplasmic reticulum in nonadrenal cells, suggesting requirement for a specific adrenal accessory factor.

C. MC3 Receptor
The MC3R gene encodes a G protein-linked receptor, coupled to both cAMP- and inositol phospholipid-Ca2+-mediated signaling systems. Polymerase chain reaction primed with degenerated oligonucleotides, whose sequence was based on the homologous transmembrane regions of the other seven transmembrane G protein-linked receptors, identified a third member of the melanocortin receptor family that recognizes the core heptapeptide sequence of melanocortins. The MC3R is the only MCR activated by ?-MSH with potency similar to that of other melanocortins (?-MSH = ACTH ? ?-MSH). This intronless gene encodes a protein of 361 amino acids.

MC3R expression occurs in brain, placenta, and gut but not in melanoma cells or in the adrenal gland. MC3R expression also occurs in the heart, in human monocytes, and in mouse peritoneal macrophages. A map of MC3R expression in the brain obtained by in situ hybridization showed abundant presence in the hypothalamus and limbic system, but signals for this receptor were also present in the septum, thalamus, hippocampus, and midbrain. POMC neurons of the rat arcuate nucleus were found to express mRNA for MC3R.

MC3R appears to participate in modulation of autonomic functions, feeding, and inflammation. Hypotension and bradycardia elicited by the release of ?-MSH from the arcuate neurons appear to be mediated by MC3R and MC4R located in the medullary dorsal-vagal complex. Participation of MC3R in energy homeostasis was disclosed in MC3R-deficient mice, which showed increased fat mass, reduced lean mass, and higher ratio of weight gain to food intake. Recent data suggest that MC3R activation mediates protective influences of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias in rats. Furthermore, activation of MC3R has clear anti-inflammatory influences.

D. MC4 Receptor
The human MC4R was the second neural MCR to be cloned. Its affinity for the melanocortins has certain similarities with that of MC1R. The order of potency for activation of MC4R is ?-MSH = ACTH > ?-MSH >> ?-MSH.
MC4R is a 332-amino acid protein encoded by a single exon of 999 nucleotides. The rat homologous gene is 93% identical to the human gene, which suggests that the gene is highly conserved in mammals.

By the use Northern blot analysis and in situ hybridization techniques, MC4R was found primarily in the brain. The distribution of this receptor in the central nervous system is much broader than that of MC3R and includes the cortex, the thalamus, the hypothalamus, the brainstem, and the spinal cord. Conversely, the MC4R was not detected in peripheral cells in an extensive study including 20 different tissues.

Distribution of MC4R is consistent with its involvement in autonomic and neuroendocrine functions. Evidence that this receptor subtype regulates food intake and energy expenditure is based on gene-targeting in mice, which results in maturity-onset obesity, with hyperphagia, hyperinsulinemia, and hyperglycinemia. Homozygous MC4R-deficient mice do not respond to the anorectic effects of ?-MSH. It appears, therefore, that ?-MSH inhibits food intake through activation of MC4R. Mice with MC4R deficiency have enhanced caloric efficiency, similar to that observed in the agouti obesity syndrome and in the MC3R-null mice. Mice lacking both MC3R and MC4R are significantly heavier than those deficient in MC4R only, suggesting that the two receptors serve nonredundant functions in the regulation of energy homeostasis. Recent research indicates that MC4R modulates erectile function and sexual behavior, possibly through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis.

E. MC5 Receptor
The melanocortin-5 receptor (MC5R) is similar to the MC1R and MC4R in its capacity to recognize ?-MSH and ACTH but not ?-MSH (?-MSH ? ACTH >> ?-MSH). MC5R contributes to regulation of exocrine gland function and to certain immune responses.
The MC5R was the last of the MCR gene family to be cloned by homology screening from genomic DNA in man, mouse, and rat. The human gene encodes for a protein of 325 amino acids.

MC5R is ubiquitously expressed in peripheral tissues. It occurs in the adrenal glands, fat cells, kidney, liver, lung, lymph nodes, bone marrow, thymus, mammary glands, testis, ovary, pituitary testis, uterus, esophagus, stomach, duodenum, skin, lung, skeletal muscle, and exocrine glands. Presence of MC5R in B- and T-lymphocytes suggests a function in immune regulation. Indeed, recent data suggest that ?-MSH participates in B-lymphocyte function via the activation of the Jak/STAT pathway, the intracellular phosphorylation pathway used by cytokines and growth factors, through specific binding to the MC5R. Furthermore, ?-MSH can induce CD25+ CD4+ regulatory T cells through the MC5R expressed on primed T cells.

Targeted disruption of the MC5R gene produced mice with a severe defect in water repulsion and thermoregulation caused by decreased production of sebaceous lipids. High expression of MC5R occurs in multiple exocrine tissues, and the receptor is required for production of porphyrins by the Harderian gland and for protein and tear secretion by the lacrimal gland. These data suggest a coordinated system for regulation of exocrine gland function by melanocortin peptides; also, that the MC5R is the mediator of the sebotrophic activity of ?-MSH described in early studies.

F. Agouti and Agouti Gene-Related Protein
Two melanocortin receptor antagonists, the agouti (also termed agouti signaling protein) and AgRP, participate in control of melanocortin signaling. Both agouti and AgRP contain cysteine-rich C-terminal domains that form disulfide bridges leading to similar folded structures. The entire antagonistic activity for MCRs resides in the Cys-rich end of the molecule. The agouti was described as a genetic locus controlling skin pigmentation long before it was cloned. In rodents, the agouti consists of a 131-amino acid protein, showing characteristics of a secreted protein with a hydrophobic signal sequence, which is expressed in skin only. The human agouti is a protein closely homologous to the rodent agouti, but it shows a much wider distribution as it is expressed in adipose tissue, testis, ovary, heart, and, at lower levels, in fore-skin, kidney, and liver. Agouti is a competitive antagonist at melanocortin receptors with high affinity at MC1R, although it also shows antagonistic activity for the human MC4R. This receptor antagonist may be important in inflammatory responses. Indeed, mice carrying the dominant agouti allele lethal yellow showed greater acute inflammatory responses than control animals.

The AgRP, a competitive antagonist for MC3R and MC4R, was cloned on the basis of its homology to agouti. The AgRP shows a very distinct expression in the central nervous system, as it is expressed in neural cell bodies of posterior hypothalamus in close vicinity to the POMC-expressing neurons. AgRP-containing neurons project to many of the same hypothalamic nuclei that receive projections from POMC neurons. The POMC and AgRP systems may function as physiologically opposing systems, where the former decreases the drive for feeding and the latter increases it.

IV. Intracellular Signaling
The transmembrane signaling of melanocortin peptides involves stimulation of adenylyl cyclase followed by synthesis of cAMP, which induces activation of protein kinase(s) and protein phosphorylation. The first report that adenylyl cyclase is involved in mediating effects of MSH appeared in 1965. Subsequently, many investigators have shown that signaling of melanocortin peptides arises via activation of adenylyl cyclase and elevation of cAMP. Stimulation of cAMP production by the MCRs causes activation of PKA, the catalytic subunit of which phosphorylates the cAMP response element-binding protein which then binds to cAMP response elements in the DNA.

Ca2+ plays a key role in MSH-receptor binding and signal transduction, since both the affinity of the ligand to the receptor and the signaling are markedly enhanced under physiological concentrations of extracellular Ca2+, relative to transduction and binding under Ca2+-free conditions. Binding of ?-MSH to B16-M2R is minimal in Ca2+-free (<50 nM) medium, reaches a first plateau between 1 and 5 ?M Ca2+, and is maximal at 1 mM Ca2+. However, although Ca2+ is required for MSH-receptor binding at low peptide concentrations (1-10 nM), it is not essential at high peptide concentrations (50-500 nM). Calmodulin inhibitors inhibit ?-MSH-receptor binding as well as the subsequent stimulation of adenylyl cyclase. This observation suggests that a calmodulin-related Ca2+-binding protein regulates binding to the receptor. Therefore, melanocortin peptides belong to the class of peptide hormones whose receptors require extracellular Ca2+ for hormone binding and signal transduction.
The affinity of MSH-peptides for their receptors is not only modulated by Ca2+ but also by GTP. Guanosine nucleotides decrease MSH-receptor binding and induce dissociation of preformed MSH-receptor complex in a calcium-independent manner.

V. Structure-Activity Relationship of Melanocortin Peptides
Basic information about structural requirements for specific functions of melanocortins was initially obtained by comparing activity profiles of naturally occurring peptides in different assays. Such comparative studies mainly evaluated melanotropic activity of each natural peptide. Subsequent research was focused on synthetic analogs and fragments of ?-MSH and other melanocortin peptides. In 1980, synthesis of 4-norleucine, 7-D-phenylalanine-?-MSH, (Nle4,D-Phe7)-?-MSH, produced a superpotent analog of the natural peptide that has been largely used since in research on ?-MSH. After recognition and cloning of melanocortin receptors, the principal aim was to design receptor-selective ligands with precise characteristics that could be useful for medical purposes. Binding assays and cAMP generation in cells transiently expressing MC1R, MC2R, MC3R, MC4R, and MC5R have improved knowledge of chemical properties that alter selectivity for each MCR subtype. Systematic amino acid substitutions were very important to design compounds that recognize specific receptor subtypes. To obtain selective compounds, it is seemingly important to identify substitutions that reduce binding for each of the receptors. Finally, mutations of receptor proteins and molecular modeling of both ligand and receptor structure have improved information on ligand binding requirements.

Over the last decade, many investigations have explored structure-activity relations of melanocortins. An inactivation study based on alanine substitutions determined the relative importance of each amino acid in the ?-MSH sequence in binding activity of ?-MSH to human MC1R and rat MC3R. This Ala-scan showed the importance of the amino acids in position 4-10 for binding to both these receptors. For binding to MC1R, Met4 appeared to be the most important amino acid outside the sequence 6-9. When this amino acid was replaced by Ala, there was a marked reduction in binding affinity for MC1R. Further investigations showed that introduction of Asp in position 4 reduced binding to all MCRs, and particularly to MC3R. His in position 6 has specific importance for binding to MC1R. Another structure-activity study focused on a tetrapeptide library, based upon the template Ac-His-D-Phe-Arg-Trp-NH2. Peptides that had been modified at the Trp9 position were characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. Results from this study showed that modification of the Trp9 in the tetrapeptide template resulted in only small changes in potency at MC1R, whereas amino acid substitutions caused up to a 9700-fold decrease in potency at MC4R and MC5R. These observations suggest that MC1R is more tolerant to modifications in the invariant sequence. Another observation from this study is that the Trp9 indole moiety in the tetrapeptide template is important for the MC3R agonist potency. This position could be used to design melanocortin ligands possessing receptor selectivity for the predominantly peripheral MC1 and MC5 relative to the centrally expressed MC3 and MC4 receptors. Indeed, potency of the Ac-His-D-Phe-Arg-Tic-NH2 and the Ac-His-D-Phe-Arg-Bip-NH2 tetrapeptides was in the nanomolar range at MC1R and MC5R but in micromolar range at MC3R and MC4R.

C-terminally modified analogs of ?-MSH indicated the importance of Pro12 for binding and activity at the MC1R. When Pro12 in the ?-MSH sequence was substituted with Phe12, the potency of the peptide was slightly reduced. Furthermore, when Phe12 was associated with Asp10, the affinity for MC1R of the peptide (Asp10, Phe12)-?-MSH was reduced to 0.069% and activity to 0.009, resulting in a virtually inactive peptide. An important observation in this research was that modifications of the melanocortin peptide sequence led to analogs for which either affinity or activity, but not both, were significantly altered. Therefore, the data confirmed the initial observations that binding to melanocortin receptors and their activation do not depend upon the same structure. Synthesis of (Nle4,D-Phe7)-?-MSH analogs where the N- or C-terminal amino acids were deleted or substituted showed that the N-terminal segment (Ser1-Tyr2-Ser3) of (Nle4,D-Phe7)-?-MSH is not important for binding to MC1R or MC4R, whereas it does influence binding to MC3R and MC5R. The C-terminal segment (Gly10-Lys11-Pro12-Val13) is important for binding to all four MCR subtypes.

The aromatic residues 1, 6, 8, and 11 and the basic residue Arg10 are the essential residues for selectivity of ?-MSH for MC3R over MC4 and MC5 receptor subtypes. A recent study shows the importance of the His-Phe-Arg-Trp sequence in receptor binding and in agonistic activity of ?-MSH. The last four amino acids in the C-terminal region of ?-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors, whereas the His-Phe-Arg-Trp sequence is relevant for activity.

Although major advances in the design and synthesis of more potent and selective MCR ligands have been made, there are still problems that need to be solved. One is that synthetic agonists or antagonists may encounter difficulties in reaching their target(s). For example, MC4R is located within the brain and, therefore, ligands must penetrate the blood-brain barrier to exert their effects. This important issue must be addressed before any new MC4R-targeted molecule could be considered for clinical use. The most widely used MC4R agonist is the cyclic lactam analog of ?-MSH Melanotan II, which penetrates the blood-brain barrier. On the other hand, reduced accessibility to central receptors could be advantageous for molecules designed to act solely in the periphery. For instance, anti-inflammatory molecules that act exclusively in the periphery should circumvent the anorectic influences of MC4R activation.

[Semin]

VI. Mechanism of the Anti-Inflammatory Action of Melanocortins
The anti-inflammatory influences of ?-MSH and other melanocortins are exerted through inhibition of inflammatory mediator production and inflammatory cell migration. These influences occur through binding of melanocortins to melanocortin receptors on immunocytes and via descending anti-inflammatory neural pathways induced by stimulation of ?-MSH receptors within the brain.

A. Receptor Subtypes Involved in the Anti-Inflammatory Effects of Melanocortins
Melanocortin peptides exert anticytokine and anti-inflammatory effects in blood cells, cells of the immune system, and in other cell types including neural, endothelial, and epithelial cells. Although these influences are mainly exerted via activation of the known melanocortin receptors, it appears that there are other, still unknown mechanisms.
A major question concerns which melanocortin receptor subtypes are involved in the anti-inflammatory influences. Virtually all the cells responsive to the anti-inflammatory effect of melanocortins express the MC1 receptor, which is the receptor with the greatest affinity for ?-MSH. This receptor subtype expression occurs in monocytes and macrophages, neutrophils, mast cells, fibroblasts, dendritic cells, astrocytes, and microglia. Thus, the MC1R likely participates in the anti-inflammatory effects of melanocortin peptides. Furthermore, it appears that MC1R expression can be altered by certain stimuli. Normal human monocytes express a small number of MC1R binding sites that are up-regulated when these cells are activated by various agents such as lipopolysaccharide (LPS) or combinations of cytokines.

Evidence from experiments using MC1R-selective synthetic analogs and from immunoneutralization studies supports the idea that MC1R activation contributes to the anti-inflammatory influences of melanocortins. Two MC1R-selective agonists, MS05 and MS09, down-regulated expression and secretion of endothelial cell selectin (E-selectin), vascular cell adhesion molecule (VCAM), and intercellular adhesion molecule (ICAM), in human dermal vascular endothelial cells treated with tumor necrosis factor ? (TNF-?). Furthermore, both MS05 and the MS09 inhibited TNF-?-induced activation of NF-?B in endothelial cells. TNF-? production was likewise reduced by the octapeptide 154N-5, which is an MC1R-specific agonist.

Experiments on immunoneutralization of the MC1R subtype in the human monocytic cell line THP-1 provide further support for the idea that the MC1R is significant in immunomodulatory effects of ?-MSH. Receptor neutralization with a specific antibody increased basal and LPS-stimulated production of TNF-? by THP-1 cells. Furthermore, preincubation of cells with the anti-MC1R antibody prevented the inhibitory influences of synthetic ?-MSH on TNF-? production. Finally, mice carrying the dominant agouti allele lethal yellow showed greater increases in circulating interleukin 6 (IL-6) after injection of LPS relative to control animals. Therefore, hypersecretion of the MC1R antagonist protein agouti enhances the acute inflammatory response to challenge.

In addition to MC1R, there is evidence that other MCR subtypes are involved in the anti-inflammatory effects of melanocortin peptides. Expression of MC3R occurs in murine and human macrophages. Natural and synthetic ligands for this receptor subtype had beneficial influences in murine urate crystal-induced peritonitis and in experimental gout. Systemic treatment with ?2-MSH in mice with urate crystal-induced peritonitis inhibited accumulation of KC/IL-8, IL-1?, and neutrophils in the peritoneal cavity. The mixed MC3/4R antagonist SHU9119 prevented the inhibitory actions of ?2-MSH, whereas the selective MC4R antagonist HS024 had no effect. Gouty arthritis induced in rats by monosodium urate monohydrate injections into rat knee joints was likewise reduced by stimulation of the MC3 receptor subtype. Finally, data from experiments on coronary ligation in rats show that MC3R participate in the protective effect of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias.

Consistent with the idea of multiple receptor involvement in the anti-inflammatory effects of melanocortins, expression of the MC5R subtype was found in human B-lymphocyte, monocyte, and mast cell lines and in lymphocytes from rat and mouse. Therefore, it appears that several MCR subtypes contribute to the anti-inflammatory effects of melanocortin peptides, perhaps in different physiological or pathological conditions, in different tissues, or at different peptide concentrations.

A still unanswered question regards the cell signaling of the C-terminal tripeptide Lys-Pro-Val, ?-MSH (11-13). This small peptide shares the anti-inflammatory and antipyretic effects of ?-MSH (1-13). Furthermore, ?-MSH (11-13) reduced NF-?B translocation to the nucleus much as the full-length ?-MSH. However, several observations indicate that this molecule does not compete with ?-MSH for receptors expressed by the B16 mouse melanoma cells and does not recognize any of the known melanocortin receptors. Therefore, the cell receptor for Lys-Pro-Val is still unknown.
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VIII. Changes in Endogenous ?-Melanocyte-Stimulating Hormone in Inflammatory Disorders
Recognition of changes in endogenous ?-MSH during localized or systemic host reactions has greatly improved understanding of the physiological role of the peptide. ?-MSH is found in plasma, and its concentration in the circulation increased after administration of pyrogens in rabbits. In normal human subjects given intravenous endotoxin, there was likewise a fever-related increase in plasma ?-MSH. Subjects with very high temperatures had marked increases in plasma ?-MSH, whereas those with lower fevers did not. These results suggest that release of ?-MSH is part of the acute phase response to infection in humans and that the molecule rapidly becomes available to modulate host responses in subjects with high fever.

Studies in human disease have shown significant changes of the endogenous peptide in pathological states. Circulating ?-MSH was increased in plasma of HIV-infected patients relative to matched controls, and its elevation was more pronounced in patients in whom the disease was more advanced. The relationship between concentrations of ?-MSH and disease outcome in HIV infection was explored in 1999. This research showed a progressive increment in plasma ?-MSH over time. The novel finding was that marked increases in concentrations of ?-MSH were associated with reduced disease progression or death, whereas patients in whom the peptide remained unchanged over time had a worse outcome. These data suggested that increased concentrations of ?-MSH have a protective influence.

Although in healthy human subjects injected with endotoxin there was a rapid increase in plasma ?-MSH, concentrations of the peptide were reduced during naturally occurring sepsis syndrome. Plasma ?-MSH was low during the critical phase of septic syndrome or septic shock and returned to normal values in patients who recovered but not in those who died. Therefore, impaired ?-MSH production had unfavorable prognostic value. Increases in circulating ?-MSH were found in patients with acute myocardial infarction and in patients on chronic hemodialysis with detectable plasma endotoxin. ?-MSH was found in the synovial fluid of adult patients with rheumatoid arthritis and young subjects with juvenile chronic arthritis. Research in children with bacteria and aseptic meningitis showed that cerebrospinal fluid concentrations of ?-MSH were elevated in those with severe bacterial meningitis with neurological sequelae.

The negative correlation between circulating ?-MSH and TNF-? early after brain injury and eventual recovery of function found in a recent research is consistent with the idea that reduced ?-MSH production in the brain after injury can have dire consequences. Indeed, the study showed marked reduction in circulating ?-MSH after acute brain injury of either traumatic or vascular origin and that patients with the lowest circulating ?-MSH had an unfavorable outcome.

These observations suggest that there is generally an increase in ?-MSH as a compensatory reaction in the presence of inflammation. It is reasonable to believe that whenever such increase does not occur or is insufficient to counteract action of inflammatory mediators, the disease process is more severe. Treatment with ?-MSH or related synthetic peptides might then be helpful for the patient.

IX. Potential Therapeutic Targets Based on Preclinical Studies in Inflammatory Disorders
Preclinical studies indicate that melanocortin peptides can be useful in treatment of localized and systemic inflammatory disorders.
A. Acute Inflammation
1. Allergic Inflammation.
Early studies on anti-inflammatory influences of ?-MSH (1-13) and (11-13) indicated that these peptides inhibit increases in capillary permeability induced by intradermal injections of histamine or IL-1 in rabbits. Subsequently, anti-inflammatory effects of ?-MSH peptides were confirmed in acute skin inflammation induced by nonspecific irritants and cytokines.

?-MSH is a significant regulatory mediator of cutaneous immune responses in vivo. When applied epicutaneously, ?-MSH inhibited both induction and elicitation of contact hypersensitivity responses in mice. Systemically administered ?-MSH likewise promoted induction of hapten-specific tolerance. Regional lymph node cells obtained from ?-MSH-treated mice after resensitization were unable to produce IL-2 in response to trinitrobenzosulfonic acid. In vivo tolerance induction by ?-MSH could be abrogated by the administration of an anti-IL-10 antibody at the site of sensitization. These data indicate that ?-MSH, in addition to its suppressive effect on induction and elicitation of contact hypersensitivity, is able to induce hapten-specific tolerance in mice and suggest that such effect is mediated by IL-10 release. Therefore contact hypersensitivity acute inflammatory reactions in the skin could be a potential therapeutic target for ?-MSH peptides.

2. Autoimmune Uveoretinitis.
Recent research indicates beneficial effects of ?-MSH in autoimmune uveoretinitis. Experimental autoimmune uveoretinitis in mice was suppressed in its severity and incidence in animals injected with primed T cells activated in vitro by antigen-presenting cells and antigen in the presence of ?-MSH. Data showed that ?-MSH converted a population of effector T cells into a population of immunoregulatory T cells. Such effector T cells showed similar TGF-?1, absence of IFN-? or IL-10, and unchanged IL-4 productions relative to inactivated T cells. These immunoregulatory influences suggest that ?-MSH could also have beneficial effects in other autoimmune disorders.

3. Gouty Arthritis.
In a model of gouty arthritis, monosodium urate monohydrate crystals were administered into rat knee joints either alone or in combination with ACTH or the selective MC3R agonist, ?2-MSH. Monosodium urate monohydrate crystals produced a knee joint inflammation that was time-dependent and characterized by white cell influx and cytokine release. Local, but not systemic, ACTH had an anti-inflammatory effect at a dose that did not alter circulating corticosterone. This treatment was also effective in adrenalectomized rats. The MC3R/MC4R antagonist SHU9119 blocked anti-inflammatory actions of ACTH but not the anti-inflammatory action of the selective MC3R agonist ?2-MSH. This research suggests that targeting MC3R subtype could be useful for clinical management of human gouty arthritis and, possibly, other acute arthritis.

B. Chronic Inflammatory Diseases
1. Rheumatoid Arthritis.
Research in patients with rheumatoid arthritis and juvenile chronic arthritis showed that ?-MSH is produced in synovial fluid of patients with these rheumatic disorders. Therefore, the peptide is likely produced at the site of inflammation to counteract influences of proinflammatory mediators. However, the local concentration of the naturally produced peptide could be inadequate to fully control the disease process. If this is true, administration of pharmacological doses of synthetic peptide could be beneficial to reduce inflammation. Consistent with this idea, treatment with ?-MSH significantly reduced joint pathology in rats with adjuvant-induced arthritis, which is a preclinical model of rheumatoid arthritis. Effectiveness of ?-MSH was similar to that of prednisolone, an established treatment for rheumatoid arthritis. However, whereas in prednisolone-treated animals and in control animals there was a significant weight reduction, ?-MSH-treated animals maintained their weight over the observation period. Therefore, in these experiments treatment with ?-MSH was very effective and devoid of the wasting effects of corticosteroids. These observations also indicate that the beneficial influences of ?-MSH on the disease course in a debilitating inflammatory disorder may prevail over the anorectic effects of the peptide. Protection against weight loss was likewise observed in a model of inflammatory bowel disease.

2. Inflammatory Bowel Diseases.
The mechanism underlying inflammatory bowel disease remains incomplete, but the importance of inflammatory processes is clear and most pharmacological therapies inhibit inflammation. Because the search for more effective agents with low toxicity continues, ?-MSH was administered to mice with dextran sulfate-induced colitis, a model of inflammatory bowel diseases. The peptide treatment had marked salutary effects; it reduced the appearance of fecal blood by over 80%, inhibited weight loss, and prevented disintegration of the general condition of the animals. Mice given ?-MSH showed markedly lower production of TNF-? by tissues of the lower colon stimulated with concanavalin A; the inhibitory effect of ?-MSH on production of inflammatory nitric oxide by lower bowel tissue was even greater.

Consistent with these observations, ?-MSH administration reduced the colonic macroscopic lesions in both acute and chronic colitis induced by trinitrobenzosulfonic acid. The salutary effect of ?-MSH on lesions of acute colitis was reversed by pretreatment with the nitric oxide donor sodium nitroprusside or the cyclooxygenase-1 selective antagonist indomethacin. The results of this study show a protective influence of ?-MSH on colonic lesions which probably involves nitric oxide and prostaglandins.

?-MSH had beneficial effects on endotoxin-induced intestinal lesions. ?-MSH treatment reduced severity of the lesions macroscopically and microscopically, although the protective effect was mainly confined to the distal ileum. The salutary effect probably involved cyclooxygenase-1 derived prostaglandins in that it was reversed by pretreatment with the nonselective cyclooxygenase-1 inhibitor indomethacin.

C. Inflammation within the Brain and Neurodegenerative Disorders
Pathogenesis of neurodegenerative disorders involves several effector molecules including cytokines, adhesion molecules, and nitric oxide. One of these molecules, TNF-?, occurs in abundance in lesions of multiple sclerosis and in other neurodegenerative disorders such as Alzheimer's disease. The capacity of TNF-? to promote myelin destruction and increased adhesion molecule expression makes it a prime suspect in etiology of neurodegeneration.

A common feature for several of the mediators involved in neurodegeneration is that their production is under control of the transcription factor NF-?B. Upon certain stimuli such as focal ischemia, glutamate, and hypoxia, there is I?B degradation and free NF-?B is translocated to the nucleus in its activated form. In research on experimental brain trauma, NF-?B was detected in the nucleus of neurons and in microglia of the injured cortex, and its activation persisted over 1 year after injury in glia of the region undergoing atrophy. NF-?B activation likewise occurred in subarachnoid hemorrhage, and inhibition of this transcription factor had beneficial influences in prevention of vasospasm. Indeed, NF-?B decoy oligo-DNA injected into the subarachnoid space inhibited cerebral vasospasm and morphological changes in vessel walls in a rabbit model for subarachnoid hemorrhage.

As stated above, ?-MSH is a potent inhibitor of NF-?B activation within the brain. Studies in experimental brain inflammation showed that ?-MSH protects the inhibitory protein I?B-? from LPS-induced degradation and reduces NF-?B activation and translocation to the nucleus. These effects were observed after either central or peripheral injections of the peptide and in vitro.

The idea that ?-MSH and other melanocortins can have protective influences in the brain is supported by the observation that ?-MSH administration prevented damage in brainstem ischemia and reperfusion injury. In research on a murine model of stroke, systemically administered ?-MSH likewise exerted neuroprotective effects in cerebral ischemia. This study showed that ?-MSH reduced intracerebral TNF-? and IL-1? gene expression after transient unilateral occlusion and reperfusion. It appears, therefore, that inhibition of NF-?B-mediated transcription by melanocortins could have beneficial influences against neurodegeneration, in particular after brain ischemia or acute injury.

Melanocortin peptides may be useful in protecting nerves from damage induced by toxins. In a rat model of cisplatin neuropathy, concomitant administration of ORG2766 protected from cisplatin-induced decrease in nerve conduction velocity. Several other preclinical studies confirmed neuroprotective influences of ORG2766 against cisplatin neurotoxicity. Furthermore, treatments with ?-MSH and ORG2766 protected against cisplatin-induced ototoxicity.
....
G. Organ Transplantation
With the increasing need for organ transplantation and the use of “marginal” organs, novel approaches are sought to increase the efficiency and survival of transplanted tissue. It was postulated that treatment with ?-MSH, which does not cause marked immunosuppression but does reduce reperfusion injury, may protect allografts and prolong their survival. NDP-?-MSH treatment caused a significant increase in allograft survival and a marked decrease in leukocyte infiltration. Expression of molecules such as endothelin 1, chemokines, and adhesion molecules, which are involved in allograft rejection, was significantly inhibited in NDP-?-MSH-treated rats. Therefore, protection of the allograft from early injury with ?-MSH can postpone rejection. Addition of this early protection with the peptide to usual treatment with immunosuppressive agents may improve success of organ transplants.

H. Infections
The rapid emergence of microorganisms resistant to conventional antibiotics has hastened the search for new antimicrobial agents. Natural antimicrobial peptides are promising candidates for treatment of resistant bacterial and fungal infections]
The C-terminal tripeptide Lys-Pro-Val, which exerts anti-inflammatory influences similar to those of the parent molecule, showed substantial candidacidal influences. A dimer of such a tripeptide, (CKPV)2, obtained by inserting a Cys-Cys linker between two units of Lys-Pro-Val-NH2, showed excellent candidacidal effects against Candida spp. including strains of C. krusei and C. glabrata. This molecule is presently in phase I/II clinical trials in the United States and in Europe and should soon be available for clinical use.

Subsequent research explored candidacidal effects of peptides derived from ?-MSH (6-13) with different amino acid substitutions. The peptide [D-Nal7,Phe12]-?-MSH (6-13), which was the most potent of the substituted peptides, reduced viability of C. albicans even more effectively than ?-MSH (1-13). ?-MSH peptides that combine antimicrobial, antipyretic, and anti-inflammatory effects could be very useful in treatment of infections.

X. Advantages over Currently Used Anti-Inflammatory Drugs and Potential Disadvantages
Preclinical investigations indicate that activation of melanocortin receptors could be a novel strategy to control inflammation. As any new therapeutic approach, this strategy may have advantages and potential disadvantages over currently available drugs. The main advantage of melanocortins in the treatment of inflammation is that their influences are broad and are not restricted to a specific mediator or chemical pathway. Indeed, as a consequence of reduced activation of the nuclear factor NF-?B, there is a collective reduction of all the major molecules involved in the inflammatory process.

Another positive feature is that treatment with melanocortin peptides never abolishes the inflammatory response; instead, it modulates it. It is current knowledge that the inflammatory response is a crucial host reaction that contributes to elimination of pathogens and harmful molecules. Cytokines, which are a significant component in the inflammatory process, also have relevant functions in regulation of tissue repair, hematopoiesis, and immune responses. Any agent that completely inhibits their production or action may have detrimental influences to the host defense. Melanocortin peptides modulate enhanced production of cytokines during infection or inflammation but do not abolish their release. Furthermore, they do not affect production of inflammatory mediators in resting conditions. A good example of this is provided by modulatory influences exerted by ?-MSH on the febrile response elicited by pyrogens without any change in nonfebrile body temperature.

A major advantage of melanocortins over currently used anti-inflammatory drugs, in particular corticosteroids, is that the peptides do not reduce microbial killing activity of neutrophils but, rather, enhance it. This characteristic could be very important to treat inflammation in an immunocompromised host.

Lack of selectivity could be a problem connected with the use of natural melanocortin peptides. It is now clear that melanocortins affect many body functions including regulation of food intake, sexual behavior, and pigmentation. Naturally produced peptides likely exert most of their effects through local paracrine and/or autocrine influences, and this may reduce influences at distant targets. Systemic injection of nonselective peptides could, therefore, cause unwanted effects through stimulation of all receptor subtypes. However, design and synthesis of new melanocortin analogs with selective affinity for specific receptors should greatly facilitate targeted effects. Knowledge of amino acid substitutions that reduce binding to each receptor can also help avoid activation of undesired receptor(s). Although recognition of each MCR function is still incomplete, there is general consensus on which receptor subtype(s) should be activated for a specific action. Indeed, current information suggests that MC1R and MC3R should be the targets for synthetic anti-inflammatory melanocortins. Furthermore, local peptide delivery at sites of inflammation, for example in the synovial fluid of an inflamed joint or into a coronary artery during reperfusion, could help convey effects to a specific target. Finally, because certain effects of the melanocortins, such as the anorectic influence, are exerted through activation of MC4R/MC3R within the brain, synthetic ligands that do or do not cross the blood-brain barrier can promote or, rather, avoid activation of these neural receptors. A synthetic anti-inflammatory peptide that does not cross the blood-brain barrier should not reduce food intake.

Another potential problem is that peptide molecules are broken down readily in the circulation or in other body fluids, and natural melanocortins are not an exception. Relatively short half-life could be problematic when sustained blood concentrations are needed. On the other hand, that peptides are not accumulated in the body reduces probability of toxicity and tolerance and allows a better control of pharmacological effects. Peptidomimetic agents targeted to melanocortin receptor subtypes could be more stable and provide sustained blood concentrations. Design and synthesis of such molecules is in progress, and they could form the basis for novel therapeutic approaches 8)

[Semin]

Chondroprotective and Anti-Inflammatory role of Melanocortin peptides in TNF-? activated human C-20/A4 chondrocytes.
2012

Abstract
Background and purpose]-?-MSH treatment also inhibited TNF-?-induced caspases 3/7 activation and chondrocyte death. The effects of [DTRP]-?-MSH, but not ?-MSH, were abrogated by the MC(3/4) receptor antagonist, SHU9119.


Conclusion and implications]Activation of MC(1) /MC(3) receptors in C-20/A4 chondrocytes down-regulates the production of pro-inflammatory cytokines and cartilage destructive proteinases, inhibits initiation of apoptotic pathways and promotes release of chondroprotective/anti-inflammatory cytokines. Developing small molecule agonists to MC(1) / MC(3) receptors could be a viable approach for developing anti-inflammatory/chondroprotective therapies in rheumatoid and osteoarthritis.