ACCELERATED COMMUNICATION [D-Trp]- -Melanocyte-Stimulating Hormone Exhibits Anti-Inflammatory Efficacy in Mice Bearing a Nonfunctional MC1R (Recessive Yellow e/e Mouse)

Two melanocortin receptors (MC1 and MC3R) have been identified as main transducers of the anti-inflammatory effects of natural and synthetic melanocortins. In this study, we have taken advantage of the recent description of the selective MC3R agonist [D-Trp]-melanocyte-stimulating hormone (MSH) and of the recessive yellow (e/e) mouse, bearing a nonfunctional MC1R, thereby incrementing our knowledge on this topic. Culturing peritoneal macrophages of recessive yellow (e/e) mice with [D-Trp]-MSH led to accumulation of cAMP, indicating MC3R receptor functionality: this effect was blocked by a neutralizing antibody against MC3R. Likewise, release of the chemokine KC by urate crystals was attenuated by [D-Trp]-MSH, and this effect was prevented by synthetic [Ac-Nlec[Asp-2 -Nal,Lys] -MSH(4–10)-NH2 (SHU9119)] and natural [agouti-related protein (AGRP)] MC3R antagonists but not by the MC4R antagonist Ac-Cys-Nle-Arg-His-D-2-Nal-Arg-TrpCys-NH2 (HS024). Systemic treatment of mice with [D-Trp ]MSH inhibited KC release and polymorphonuclear cell accumulation elicited by urate crystals in the murine peritoneal cavity. SHU9119 and AGRP prevented the inhibitory actions of [D-Trp]-MSH, whereas HS024 was inactive. We also demonstrate here that [D-Trp]-MSH displays a dual mechanism of action by inducing the anti-inflammatory protein heme-oxygenase 1 (HO-1). Treatment with the HO-1 inhibitor zinc protoporphyrin IX exacerbated the inflammatory response elicited by urate crystals and abrogated the anti-inflammatory effects of [D-Trp]-MSH. In conclusion, these data support the development of the selective MC3R agonist [D-Trp]-MSH for the treatment of inflammatory pathologies, based on a dual mechanism of cytokine/chemokine inhibition and induction of the anti-inflammatory protein HO-1. Melanocortins [ , , and -melanocyte-stimulating hormone (MSH)] are peptides derived from a larger precursor molecule known as the pro-opiomelanocortin gene product and are little changed throughout evolution; they are traceable back to the appearance of the first vertebrates (Lipton and Catania, 1997). They exert their numerous biological effects by activating seven-transmembrane G-protein-coupled receptors, leading to adenyl cyclase activation and subsequent increases in intracellular cAMP accumulation (Wikberg et al., 2000). The era of molecular biology has enabled identification and subsequent cloning of five melanocortin receptors. On the one hand, this has greatly improved the understanding of the potential biological roles for melanocortins; on the other hand, it has stimulated the classification for activity of compounds associated with each receptor. Melanocortin receptors have been shown to have a wide Research was funded by Research Advisory Board, St. Bartholomew’s and the Royal London School of Medicine and Dentistry (grant RAB04/PJ/04) and by the Arthritis Research Campaign UK (grant 17299). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.028878. ABBREVIATIONS: MSH, melanocyte-stimulating hormone; MØ, macrophage; HO-1, heme-oxygenase 1; MSU, monosodium urate; PBS, phosphate-buffered saline; ZnPPIX, zinc protoporphyrin IX; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; SHU9119, Ac-Nlec[Asp-2 -Nal,Lys] -MSH(4–10)-NH2; AGRP, agouti-related peptide; MTII, melanotan II; HS024, Ac-Cys-Nle-Arg-His-D-2-Nal-Arg-Trp-CysNH2. 0026-895X/06/7006-1850–1855$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 6 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 28878/3153772 Mol Pharmacol 70:1850–1855, 2006 Printed in U.S.A. 1850 at A PE T Jornals on Jne 2, 2017 m oharm .aspeurnals.org D ow nladed from and varied distribution and are found in the central nervous system, periphery, and immune cells (Catania et al., 2004). Over the last 2 decades, the pharmacological definition of each receptor has begun to be unraveled. MC1R is expressed on melanocytes and controls skin pigmentation (AbdelMalek, 2001). MC2R is expressed on adrenocortical cells (Penhoat et al., 1989) and is involved in adrenal steroidogenesis. MC3R controls macrophage (MØ) activity (Getting, 2002), MC4R is involved in the control of obesity (Ellacott and Cone, 2004) and erectile dysfunction (Martin and MacIntyre, 2004); although MC5R has not received as much attention, it seems to be involved in the control of exocrine secretion and may also have some immunoregulatory functions (Wikberg et al., 2000). These endogenous peptides have long been reported to be beneficial in many experimental inflammatory diseases including gouty arthritis (Getting et al., 1999, 2002), adjuvantinduced arthritis (Ceriani et al., 1994), inflammatory bowel disease (Rajora et al., 1997), and asthma (Raap et al., 2003). The beneficial effects of the melanocortins seem to be due to their ability to prevent nuclear factor B activation (Manna and Aggarwal, 1998; Kalden et al., 1999) via the protection of I B (Ichiyama et al., 1999), thus leading to a reduction of pro-inflammatory mediator synthesis (including cytokines) and adhesion molecule expression with the subsequent inhibition of pro-inflammatory cell migration. Studies from our group have highlighted the role played by the MC3R in inhibiting urate crystal induced inflammation (Getting et al., 1999). The naturally occurring MC3R agonist 2-MSH (Roselli-Rehfuss et al., 1993) and the synthetic peptide MTII (Hruby et al., 1995) possess anti-inflammatory efficacy in a urate model of crystal-induced peritonitis (Getting et al., 2001) and rat knee joint inflammation (Getting et al., 2002). The cellular target for these actions seems to be the MØ; MC3R is the receptor predominantly responsible for mediating them (Getting et al., 1999, 2001, 2002). More recently, we have substantiated the theory that melanocortins inhibit cytokine release ( 2 h) from MØ and, at later time points, ( 4 h) induce the anti-inflammatory protein hemeoxygenase 1 (HO-1) (Lam et al., 2005). The present study used both in vitro and in vivo pharmacological and genetic approaches to assess the anti-inflammatory efficacy of the selective MC3R agonist [D-Trp]MSH (Grieco et al., 2000). We report that [D-Trp]-MSH displays anti-inflammatory efficacy in mice bearing a nonfunctional MC1R (recessive yellow e/e mouse) and that these effects are abrogated in the presence of the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX). Materials and Methods