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Divisions of Internal Medicine (A.C., G.C.) and Liver Transplantation (S.G.), Ospedale Maggiore di Milano, Istituto di Ricovero e Cura a Carattere Scientifico, Milano, Italy; and Zengen, Inc. (J.M.L.), Woodland Hills, California
Abstract
Abstract I. Introduction II. Proopiomelanocortin Gene, Gene Expression, and Post-Translational Processing A. Proopiomelanocortin Gene B. Proopiomelanocortin Gene Expression C. Post-Translational Processing of Proopiomelanocortin D. Melanocortin Peptides III. Melanocortin Receptors and Their Endogenous Antagonists A. MC1 Receptor B. MC2 Receptor C. MC3 Receptor D. MC4 Receptor E. MC5 Receptor F. Agouti and Agouti Gene-Related Protein IV. Intracellular Signaling V. Structure-Activity Relationship of Melanocortin Peptides VI. Mechanism of the Anti-Inflammatory Action of Melanocortins A. Receptor Subtypes Involved in the Anti-Inflammatory Effects of Melanocortins B. Influence of Melanocortins on Nuclear Factor-{kappa}B-Mediated Transcription C. Melanocortins Modulate Production of Chemical Mediators of Inflammation 1. Effects in Vitro. 2. Effects in Vivo. D. Central Control of Peripheral Inflammation VII. Antipyretic Influences of Melanocortins VIII. Changes in Endogenous {alpha}-Melanocyte-Stimulating Hormone in Inflammatory Disorders IX. Potential Therapeutic Targets Based on Preclinical Studies in Inflammatory Disorders A. Acute Inflammation 1. Allergic Inflammation. 2. Autoimmune Uveoretinitis. 3. Gouty Arthritis. B. Chronic Inflammatory Diseases 1. Rheumatoid Arthritis. 2. Inflammatory Bowel Diseases. C. Inflammation within the Brain and Neurodegenerative Disorders D. Peripheral Neuropathies E. Systemic Host Reactions 1. Septic Shock. 2. Systemic Vasculitis. 3. Acute Respiratory Distress Syndrome. 4. Hemorrhagic Shock. F. Ischemia and Reperfusion Injury G. Organ Transplantation H. Infections X. Advantages over Currently Used Anti-Inflammatory Drugs and Potential Disadvantages
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 already in progress. This review examines how marshaling MCR could control inflammation.
Adrenocorticotropic hormone (ACTH) and -, -, and -melanocyte-stimulating (-, -, -MSH) hormones derive from post-translational processing of the precursor molecule proopiomelanocortin (POMC) (Eberle, 1988
; Hadley and Haskell-Luevano, 1999) (Fig. 1). 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 over 50 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.
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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 (Beck, 2000; Wessells et al., 2000; Adan and Vink, 2001; Boston, 2001; Crowley et al., 2002; MacNeil et al., 2002; Van der Ploeg et al., 2002; Goodfellow and Saunders, 2003). A detailed description of effects of melanocortins in skin physiology and melanocyte function (Luger et al., 1997; Hadley et al., 1998; Slominski and Pawelek, 1998; Thody, 1999; Abdel-Malek et al., 2000; Bohm and Luger, 2000; Slominski and Wortsman, 2000; Slominski et al., 2000) and influences on behavior, learning, and memory (Beckwith et al., 1977, 1989; Datta and King, 1982; Klusa et al., 1998; Smolnik et al., 2000) are likewise beyond the scope of this review.
II. Proopiomelanocortin Gene, Gene Expression, and Post-Translational Processing
In 1977, a common precursor for ACTH/-MSH and -lipotropin (-LPH)/-MSH/-endorphin was found in mouse AtT-20 pituitary tumor cells (Mains et al., 1977; Roberts and Herbert, 1977). One year later, the molecule was demonstrated in a human nonpituitary tumor cell line (Bertagna et al., 1978). In 1979, analysis of the nucleotide sequence of cloned cDNA of bovine POMC (Nakanishi et al., 1979) showed a hitherto unknown MSH-like peptide sequence, called -MSH, as well as other N-terminal peptides. Gene sequences for human (Chang et al., 1980) and rat (Drouin and Goodman, 1980) 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 (Heinig et al., 1995). In humans, the POMC gene is unusual in that it possesses three promoter regions that control transcription: promoter 1 (P1), P2, and P3 (Kraus et al., 1993). P1 is the predominant promoter in some cancers; P2 controls transcription in the normal pituitary gland; and P3 is weakly active in a variety of peripheral tissues (Kraus et al., 1993).
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 (Cone et al., 2001; Pritchard et al., 2002). POMC mRNA is also detectable in the spinal cord and dorsal root ganglion (Plantinga et al., 1992; van der Kraan et al., 1999). 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 (Bagnol et al., 1999). The POMC neurons in the arcuate nucleus express leptin receptors, through which leptin regulates POMC expression (Schwartz et al., 1997; Elmquist et al., 1999). 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 (Broberger et al., 1998; Bagnol et al., 1999). 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 (Beuschlein and Hammer, 2002) knowledge that POMC is expressed also in normal tissues is more recent (Blalock, 1985, 1999). 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 (Star et al., 1995; Chakraborty et al., 1996; Blalock, 1999; Slominski and Wortsman, 2000; Slominski et al., 2000). There is evidence that leukemia inhibitory factor stimulates POMC expression via phosphorylation of signal transducers and activators of transcription (STATs) STAT1 and STAT3 proteins (Ray et al., 1996). 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 (Farooqui et al., 1995; Millington et al., 1999).
C. Post-Translational Processing of Proopiomelanocortin
Biologically active proteins and peptides are often generated by intracellular proteolysis of inactive precursors (Seidah et al., 1999). 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 (Seidah et al., 1991). Thus, generation of biologically active peptides and proteins depends on two main components: the polypeptide precursor substrate and the proteolytic enzyme(s) responsible for the conversion of the precursor into its final bioactive protein-peptide product.
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 (Seidah and Chretien, 1994). There are seven known mammalian precursor (or proprotein) convertases (PCs) cleaving at single and/or pairs of basic residues: PC1 (also called PC3), PC2, furin (also called PACE), PACE4, PC4, PC5 (also called PC6), and PC7 (also called SPC7, LPC, or PC8) (Seidah and Chretien, 1994; Rouillé et al., 1995; Seidah et al., 1999). PCs show a remarkable temporal and spatial specificity of expression patterns that make them available in various proportions and combinations in different cell types. Conservation of the catalytic domains and variability of these domains suggest that PC genes evolved from a common ancestral gene. Except for furin and PACE4, which are closely linked, all the PC genes are dispersed on various chromosomes. Recent research indicates that although many PCs could cleave the same precursor in vitro, in vivo processing depends on regulation of individual cellular expression and activity as well as intraorganellar localization, which are critical for each processing reaction (Seidah et al., 1999).
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 (Malide et al., 1995). 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 (Seidah et al., 1992, 1996; Seidah and Chretien, 1994). 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 (Benjannet et al., 1991). 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 (Day et al., 1992; Marcinkiewicz et al., 1993). 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 (Blalock, 1985) and the skin (Wakamatsu et al., 1997).
Amino acid sequences of human melanocortin peptides are shown in Fig. 2. - and -MSH and ACTH were purified and sequenced in the 1950s (Eberle, 1988). -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 (Eberle, 1988). -MSH was originally isolated from human pituitaries as a side fraction of somatotropin preparation (Dixon, 1960) and thereafter found to correspond to the 37-58 region of human -LPH (Li and Chung, 1976). 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 (Bertagna et al., 1986). 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.
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III. 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 (Cone, 2000). 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 (Fig. 3). All are functionally coupled to adenylyl cyclase and mediate their effects primarily by activating a cAMP-dependent signaling pathway.
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MCRs share many features with other G protein-coupled receptors (Rana, 2003): they have several potential N-glycosylation sites in their amino-terminal domains, consensus recognition sites for protein kinase C and/or A (PKA), and conserved cysteines in their carboxyl termini (Wikberg et al., 2000; Abdel-Malek, 2001). Ligand affinity, tissue distribution, and functions of each receptor subtype are reported in Table 1.
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MC1R was the first member of the MCR gene family to be cloned (Chhajlani and Wikberg, 1992; Mountjoy et al., 1992). 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 (Chhajlani and Wikberg, 1992; Suzuki et al., 1996). 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 (Abdel-Malek, 2001).
-MSH/MC1R interactions contribute to regulation of skin physiology and melanogenesis (Slominski et al., 2000). 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 (Hunt et al., 1994; Abdel-Malek et al., 1995). MC1R mRNA in the skin is up-regulated by its own melanocortin ligands and by endothelin-1 (Abdel-Malek, 2001). 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 (Smith et al., 2001), in coexpression studies, induction of MC1R promoter activity increased by 5-fold in the presence of MITF (Aoki and Moro, 2002). In addition to effects in melanocytes, there is evidence that MITF enhances expression of MC1R in cultured murine mast cells (Adachi et al., 2000).
It is clear that MC1R functions extend well beyond regulation of melanogenesis. MC1R expression occurs in macrophage/monocytic cells (Star et al., 1995; Bhardwaj et al., 1997; Taherzadeh et al., 1999), lymphocytes with antigen-presenting and cytotoxic functions (Neumann Andersen et al., 2001), neutrophils (Catania et al., 1996), endothelial cells (Hartmeyer et al., 1997), astrocytes (Wong et al., 1997), and fibroblasts (Bohm et al., 1999b). Peripheral blood-derived dendritic cells were likewise found to express MC1R (Becher et al., 1999; Salazar-Onfray et al., 2002). 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 (Xia et al., 1995). Transactivation of MC1R in inflammatory cells causes marked reduction of activation and translocation to the nucleus of the transcription factor NF-B (Manna and Aggarwal, 1998). Consequently, there are marked anti-inflammatory effects exerted through inhibition of NF-B-mediated transcription (see below).
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 (Suzuki et al., 1996; Kalden et al., 1999; Scholzen et al., 1999). Therefore, although receptor density in inflammatory cells is less than that in melanocytes, it appears that receptor affinity is much greater.
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 (Lefkowitz et al., 1970; Buckley and Ramachandran, 1981; Mountjoy et al., 1992). 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 (Penhoat et al., 1989).
In situ hybridization studies revealed dense expression of MC2R (Mountjoy et al., 1992; Xia and Wikberg, 1996) in the zona glomerulosa and zona fasciculata of the adrenal cortex, the sites of mineralocorticoid and glucocorticoid production (Mountjoy et al., 1992; Liakos et al., 1998). The zona reticularis showed less mRNA labeling. In the adrenal medulla, only scattered cells with unknown functions stain for MC2R mRNA (Xia and Wikberg, 1996). MC2R expression in adrenal cells is up-regulated by its ligand ACTH (Mountjoy et al., 1994a).
In addition to the adrenal glands, the MC2R mRNA has been found in murine adipocytes (Boston and Cone, 1996; Cammas et al., 1997), where it is believed to mediate stress-induced lipolysis in response to ACTH (Boston, 1999). However, ACTH does not appear to regulate adipocyte function in humans and other primates, as human adipocytes lack expression of MC2R (Chhajlani, 1996). 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 (Noon et al., 2002).
The MC3R gene encodes a G protein-linked receptor, coupled to both cAMP- and inositol phospholipid-Ca2+-mediated signaling systems (Konda et al., 1994). 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 (Gantz et al., 1993a). The MC3R is the only MCR activated by -MSH with potency similar to that of other melanocortins (-MSH = ACTH -MSH) (Roselli-Rehfuss et al., 1993). 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 (Gantz et al., 1993a). MC3R expression also occurs in the heart (Chhajlani, 1996), in human monocytes (Taherzadeh et al., 1999), and in mouse peritoneal macrophages (Getting et al., 1999). 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 (Roselli-Rehfuss et al., 1993). POMC neurons of the rat arcuate nucleus were found to express mRNA for MC3R (Jegou et al., 2000).
MC3R appears to participate in modulation of autonomic functions, feeding, and inflammation (Abdel-Malek, 2001; Getting, 2002). 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 (Li et al., 1996). 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 (Chen et al., 2000). Recent data suggest that MC3R activation mediates protective influences of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias in rats (Guarini et al., 2002). Furthermore, activation of MC3R has clear anti-inflammatory influences (Getting et al., 1999, 2002, 2003a; Getting and Perretti, 2000).
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 (Alvaro et al., 1996), 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 (Gantz et al., 1993b). 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 (Mountjoy et al., 1994b). Conversely, the MC4R was not detected in peripheral cells in an extensive study including 20 different tissues (Chhajlani, 1996).
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 (Huszar et al., 1997). 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 (Marsh et al., 1999). Mice with MC4R deficiency have enhanced caloric efficiency, similar to that observed in the agouti obesity syndrome and in the MC3R-null mice (Ste Marie et al., 2000). 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 (Chen et al., 2000). 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 (Wessells et al., 1998; Van der Ploeg et al., 2002).
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 (Chhajlani et al., 1993), mouse (Labbe et al., 1994), and rat (Griffon et al., 1994). 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 (Gantz et al., 1994; Labbe et al., 1994; Fathi et al., 1995; Chhajlani, 1996; Chen et al., 1997; van der Kraan et al., 1998; Colombo et al., 2002). 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 (Buggy, 1998). Furthermore, -MSH can induce CD25+ CD4+ regulatory T cells through the MC5R expressed on primed T cells (Taylor and Namba, 2001).
Targeted disruption of the MC5R gene produced mice with a severe defect in water repulsion and thermoregulation caused by decreased production of sebaceous lipids (Chen et al., 1997). 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 (Entwistle et al., 1990; Chen et al., 1997). 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 (Thody and Shuster, 1970, 1975).
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 (Ollmann et al., 1998; Wikberg et al., 2000). Both agouti and AgRP contain cysteine-rich C-terminal domains that form disulfide bridges leading to similar folded structures (Dinulescu and Cone, 2000). The entire antagonistic activity for MCRs resides in the Cys-rich end of the molecule (Willard et al., 1995). The agouti was described as a genetic locus controlling skin pigmentation long before it was cloned (Seechurn et al., 1988). 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 (Bultman et al., 1992; Lu et al., 1994). 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 (Wilson et al., 1995; Voisey and van Daal, 2002). Agouti is a competitive antagonist at melanocortin receptors with high affinity at MC1R (Blanchard et al., 1995), 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 (Lipton et al., 1999).
The AgRP, a competitive antagonist for MC3R and MC4R, was cloned on the basis of its homology to agouti (Ollmann et al., 1997). 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 (Broberger et al., 1998). 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 (Wilson et al., 1999; Wirth and Giraudo, 2000).
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 (Eberle et al., 1978; Eberle, 1988). The first report that adenylyl cyclase is involved in mediating effects of MSH appeared in 1965 (Bitensky and Burstein, 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 (Busca and Ballotti, 2000; Harris et al., 2001; Sarkar et al., 2002).
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 (Gerst et al., 1987). Binding of -MSH to B16-M2R is minimal in Ca2+-free (<50 nM) medium, reaches a first plateau between 1 and 5 µM 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 (Gerst and Salomon, 1988). 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 (Rodbell, 1980; Gerst et al., 1987).
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 (Hruby et al., 1987; Eberle, 1988). 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 (Sawyer et al., 1980). 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 (Haskell-Luevano et al., 1996; Schioth et al., 1997a, 2002; Wikberg et al., 2000; Grieco et al., 2002; Kavarana et al., 2002; Han et al., 2003). 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 (Haskell-Luevano et al., 1996; Yang et al., 1997; Prusis et al., 2001; Holder and Haskell-Luevano, 2003).
Over the last decade, many investigations have explored structure-activity relations of melanocortins (Hruby and Han, 2000). 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 (Sahm et al., 1994). This Ala-scan showed the importance of the amino acids in position 4-10 for binding to both these receptors (Sahm et al., 1994). 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 (Sahm et al., 1994). Further investigations showed that introduction of Asp in position 4 reduced binding to all MCRs, and particularly to MC3R (Schioth et al., 2002). His in position 6 has specific importance for binding to MC1R (Sahm et al., 1994; Schioth et al., 1997b, 2002). 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 (Holder et al., 2002). 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 (Holder et al., 2002).
C-terminally modified analogs of -MSH indicated the importance of Pro12 for binding and activity at the MC1R (Peng et al., 1997). 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 (Peng et al., 1997). Therefore, the data confirmed the initial observations that binding to melanocortin receptors and their activation do not depend upon the same structure (Eberle, 1988). 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 (Schioth et al., 1998). 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 (Grieco et al., 2000). A recent study shows the importance of the His-Phe-Arg-Trp sequence in receptor binding and in agonistic activity of -MSH (Grieco et al., 2002). 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 (Al-Obeidi et al., 1989). 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.
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 (Table 2). 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 (Lipton and Catania, 1997).
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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 (de Angelis et al., 1995; Star et al., 1995; Catania et al., 1996; Rajora et al., 1996; Hartmeyer et al., 1997; Artuc et al., 1999; Becher et al., 1999; Bohm et al., 1999a; Chakraborty et al., 1999; Luger et al., 2000; Scott et al., 2002). 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 (Bhardwaj et al., 1997).
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 (Szardenings et al., 2000), 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-) (Brzoska et al., 1999a). 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 (Ignar et al., 2003).
Experiments on immunoneutralization of the MC1R subtype in the human monocytic cell line THP-1 (Taherzadeh et al., 1999) 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 (Taherzadeh et al., 1999). 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 (Lipton et al., 1999). 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 (Getting et al., 1999, 2001) and human (Taherzadeh et al., 1999) macrophages. Natural and synthetic ligands for this receptor subtype had beneficial influences in murine urate crystal-induced peritonitis and in experimental gout (Getting et al., 2001, 2002). 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 (Getting et al., 2001). The mixed MC3/4R antagonist SHU9119 (Hruby et al., 1995) prevented the inhibitory actions of 2-MSH, whereas the selective MC4R antagonist HS024 (Kask et al., 1998) 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 (Getting et al., 2002). 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 (Guarini et al., 2002).
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 (Buggy, 1998), monocyte (Taherzadeh et al., 1999), and mast cell (Artuc et al., 1999) lines and in lymphocytes from rat (Akbulut et al., 2001) and mouse (Taylor and Namba, 2001). 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) (Richards and Lipton, 1984b; Hiltz and Lipton, 1989; Mugridge et al., 1991; Hiltz et al., 1992; Poole et al., 1992; Uehara et al., 1992; Watanabe et al., 1993; Ceriani et al., 1994b; Macaluso et al., 1994; Bhardwaj et al., 1996; Ichiyama et al., 1999c; Luger et al., 1999; Haddad et al., 2001; Mandrika et al., 2001). Furthermore, -MSH (11-13) reduced NF-B translocation to the nucleus much as the full-length -MSH (Lipton et al., 1999; Barcellini et al., 2000; Mandrika et al., 2001). However, several observations indicate that this molecule does not compete with -MSH for receptors expressed by the B16 mouse melanoma cells (Lyson et al., 1994) and does not recognize any of the known melanocortin receptors (Wikberg et al., 2000; Mandrika et al., 2001; Getting et al., 2003b; Muceniece et al., 2003). Therefore, the cell receptor for Lys-Pro-Val is still unknown.
B. Influence of Melanocortins on Nuclear Factor-B-Mediated Transcription
The remarkably broad effects of -MSH on inflammatory mediator production were puzzling to researchers until the discovery that the peptide inhibits activation of the nuclear factor-B (Manna and Aggarwal, 1998; Haycock et al., 1999, 2000; Ichiyama et al., 1999a,b,c,d, 2000b; Gupta et al., 2000; Haddad et al., 2001; Mandrika et al., 2001; Hassoun et al., 2002). This essential nuclear factor induces transcription of many molecules involved in the inflammatory process: its inhibition has, therefore, broad consequences for mediator production and cell functions. NF-B is present in virtually all eukaryotic cell types. It consists of homodimers and heterodimers of proteins of the Rel family. The first member of this family to be described and the factor commonly referred to as NF-B consists of a heterodimer of p50 and p65 subunits. NF-B is retained in an inactive form in the cytoplasm bound to members of the IB inhibitory protein family (May and Ghosh, 1998). Phosphorylation of IB by various agents such as drugs, cytokines, bacterial products, and viruses can cause IB degradation. Subsequently, the free NF-B is translocated to the nucleus where it binds to sequences of DNA encoding NF-B-responsive elements and triggers the transcription of target genes. NF-B participates in the regulation of hundreds of genes, including those for cytokines, chemokines, growth factors of the hematopoietic system, major histocompatibility system, antiapoptotic factors, and inducible nitric oxide synthase (iNOS). Therefore, the discovery that -MSH inhibits NF-B activation has provided an explanation for the broad effects of the peptide on mediator production. In the monocytic cell line U937, -MSH down-regulated NF-B activation induced by a variety of inflammatory stimuli including TNF, endotoxin, ceramide, and okadaic acid (Manna and Aggarwal, 1998). Suppression of NF-B translocation occurred through generation of cAMP and activation of PKA (Manna and Aggarwal, 1998). Similar results were obtained in experiments on human glioma cells and whole mouse brains stimulated with lipopolysaccharide. -MSH and its C-terminal tripeptide KPV modulated brain inflammation by inhibiting NF-B activation (Ichiyama et al., 1999d). In both models of central nervous system inflammation, the evidence was consistent with -MSH-induced modulation of NF-B activation by limiting IB- degradation. Furthermore, -MSH modulated activation of NF-B in human dermal fibroblasts (Bohm et al., 1999b), endothelial cells (Kalden et al., 1999), keratinocytes (Brzoska et al., 1999b), melanocytes (Haycock et al., 1999), and melanoma cells (Haycock et al., 1999).
Experiments on cells transfected with a plasmid vector encoding -MSH indicate that the peptide can inhibit NF-B activation in an autocrine fashion (Ichiyama et al., 1999a, 2000b). In glioma cells and lung epithelial cells transfected with pCMV-ssMSH, there was reduced I-B degradation and inhibition of NF-B activation (Ichiyama et al., 1999a, 2000b). Inhibition of NF-B by plasmid-expressed -MSH was likewise observed in human fetal kidney and cytoskeletal muscle cells (Etemad-Moghadam et al., 2002).
Recent research indicates that -MSH inhibits NF-B activation also in human immunodeficiency virus (HIV)-infected monocytes (Barcellini et al., 2000). Through binding of its long terminal repeat to NF-B, HIV replication is linked to the state of activation of infected cells. Stimuli that activate NF-B enhance HIV production (Feinberg, 1992). In these circumstances, viral RNA increases, and the pattern of expression changes to include the singly spliced and unspliced messenger RNA transcripts encoding virion constituents. -MSH reduced NF-B activation and spliced and unspliced HIV RNA in phorbol 12-myristate 13-acetate-stimulated chronically HIV-infected U1 cells (Barcellini et al., 2000). All these observations suggest that -MSH could be a candidate for treatment of pathologic conditions in which activation of NF-B is prominent.
C. Melanocortins Modulate Production of Chemical Mediators of Inflammation
Since the initial studies on antipyretic and anti-inflammatory influences of -MSH, it has been clear that the peptide inhibits production and action of cytokines and other mediators of inflammation (Catania and Lipton, 1993). Inhibitory effects on inflammatory mediator production were observed in experiments in vitro and in vivo. In vitro experiments indicated anti-inflammatory influences both in normal cells and in cells from blood of patients with inflammatory or infectious disorders. Effects in vivo occurred during systemic or localized inflammatory host challenge.
1. Effects in Vitro.
Monocytic cells are significant targets for the anti-inflammatory effects of -MSH. -MSH down-regulated CD86, a major T-cell costimulatory molecule, in LPS-stimulated monocytes (Bhardwaj et al., 1997). In human peripheral blood monocytes and cultured human monocytes, -MSH increased the production and expression of IL-10 (Bhardwaj et al., 1996). Because IL-10 reduces proinflammatory cytokine production in macrophages, its up-regulation can have anti-inflammatory influences. Research in septic patients showed that addition of small concentrations of -MSH to LPS-stimulated whole blood samples inhibited TNF- and IL-1 production by 30 to 40% (Catania et al., 2000b). Cytokine production in monocytes was inhibited also when production was stimulated by a noninflammatory inducer. Indeed, in experiments on normal peripheral blood mononuclear cells, -MSH inhibited the production of IL-1 and TNF- induced by HIV envelope glycoprotein 120 (gp120) (Catania et al., 1998b). The inhibitory effect of -MSH on TNF- production was also observed in whole blood from HIV-positive patients stimulated with endotoxin (Catania et al., 1998b).
The inhibitory effects of -MSH on prod
