Melanotan II possess anti-inflammatory response
Melanocortin Melanotan II possess anti-inflammatory response
« on: November 16, 2010, 10
11 AM »In Vitro and In Vivo Induction of Heme Oxygenase 1 in Mouse Macrophages following Melanocortin Receptor Activation
Connie W. Lam, Stephen J. Getting and Mauro Perretti2
The William Harvey Research Institute, Bart’s and the London, Queen Mary School of Medicine and Dentistry, London, United Kingdom
Abstract
RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4–24 h) and concentration-related (1–100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (M{phi}) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary M{phi}. HO-1 induction in peritoneal M{phi} was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the M{phi}, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.
Introduction
Melanocortin peptides (e.g., {alpha}-melanocortin-stimulating hormone) have long been reported to possess anti-inflammatory effects in many experimental models of acute and chronic inflammation, including inflammatory bowel disease (3), allergy (4), joint arthritis (5, 6), and systemic inflammation (endotoxemia) (7). Interestingly, recent trials with {alpha}-MSH have confirmed the positive indication for this compound in controlling human disease (Cool, thereby reinforcing the potential impact of this line of research. Because {alpha}-MSH represents the first 13 aa within the adrenocorticotrophin (ACTH) sequence (39 aa in total) (9), it is important to recall the efficacy of the longer polypeptide in controlling rheumatoid arthritis (10).
At the molecular level, the effects of these anti-inflammatory hormones and synthetic derivatives on target cells are brought about by activation of a subgroup of G protein-coupled receptor, termed melanocortin receptors (MC-R). Five MC-Rs have been identified so far, and all of these receptors are positively coupled to adenylate cyclase such that their activation leads to increases in intracellular cAMP (9, 11). Following this early event of cAMP formation, and also perhaps partly independently from it, melanocortin peptides have been shown to down-regulate NF-{kappa}B activation and consequent cytokine synthesis (12, 13). The C terminus sequence (i.e., aa 11–13) of {alpha}-MSH outside the common core and modifications of it (14, 15) have been shown to block cytokine functions (1), rather than synthesis and release (16).
Heme oxygenase (HO)-1 is the rate-limiting enzyme in heme catabolism with consequent generation of biliverdin (then converted to bilirubin), free iron, and carbon monoxide. Three mammalian HO isoforms have been identified, one of which, HO-1, is a stress responsive protein endowed with important cytoprotective effects (for a recent review, see Ref.17). In addition, macrophage (M{phi}) HO-1 expression is part of the repairing processes that occur during resolving inflammation leading to healing and tissue repair (18). It is possible that at least some of the cytoprotective and anti-inflammatory actions of HO-1 are due to the controlled local liberation of carbon monoxide, able to signal through the cyclic GMP pathway (17) and inhibit cytokine synthesis (19). In addition, the other catabolite bilirubin is also endowed with antioxidant and anti-inflammatory effects, for instance, its application inhibits LPS-induced selectin expression in the vasculature, thus affecting leukocyte recruitment (20).
The present study was undertaken to assess a potential functional link between MC-R-dependent cAMP formation and HO-1 induction in M{phi}. Most of the experiments have been conducted with the RAW264.7 M{phi} cell line, both for data consistency and ease of manipulation (and reduction in animal sacrifice); however, crucial experiments have been repeated with primary M{phi}. Importantly, in vivo experimentation not only confirmed the biochemical link between MC-R activation and HO-1 induction, but also provided a functional relevance to this interaction. We conclude that MC3-R activation, and possibly activation of other MC-R subtype as well, can bring about anti-inflammatory effects mediated, at least in part, by HO-1 induction.
Discussion
This study demonstrates a previously unknown link between M{phi} MC3-R activation and induction of the anti-inflammatory enzyme HO-1. Activation of this receptor by MTII, and the less selective agonist ACTH, produces transient alterations in intracellular cAMP that are temporally related to HO-1 up-regulation in a PKA-dependent fashion. In vivo, the MC3-R/HO-1 connection is functionally operative in bringing about the antimigratory effect of a melanocortin peptide.
Since the initial studies with {alpha}-MSH (27), melanocortin peptides have been shown to represent an important component of the counterregulatory systems that operate in the host to dampen and control the inflammatory reaction. Several studies conducted in experimental animals with models of acute and subacute inflammation (1, 28), in some cases supported by human data (29), have shown how {alpha}-MSH and other melanocortin peptides are endowed with potent inhibitory and anti-inflammatory properties (Cool. This field attracted much more interest once specific receptors were cloned and shown to mediate the actions of several melanocortins, including the naturally occurring ACTH and {alpha}-MSH, on different target cells. MC-R belongs to the family of G protein-coupled receptors, and their activation leads to adenylate cyclase-mediated conversion of ATP into cAMP (9, 11). Thus, accumulation of cAMP in target cells buffers cell activation with a marked effect on the production of proinflammatory cytokines (12, 13, 21). Therefore, targeting specific MC-R could certainly represent a novel strategy to develop innovative anti-inflammatory agents, as recently reviewed (30).
The similarity in mediated signaling events for the five MC-R can be problematic for drug development, impeding exploitation of specific postreceptor pathways: this aspect is made more acute by the absence of specific pharmacological tools to dissect the functions of each receptor. A few years ago, we started a project focusing on the resident M{phi}, reasoning that a certain degree of specificity could derive from narrowing down actions of a given target cell. Addition of ACTH, {alpha}-MSH, and the relatively more specific synthetic derivative MTII (31), to mouse peritoneal M{phi} inhibited the release of an array of cytokines and chemokines (21, 22). Comparison of the effects produced by agonists and antagonists, together with morphological analyses and study with mutated receptors, allowed us to pinpoint MC3-R as a major determinant for these inhibitory properties, at least at the level of the M{phi} (6).
MC3-R-mediated inhibition of cytokine synthesis and release from stimulated M{phi} in vitro occurs relatively rapid (≤2 h). In the present study, we set out to examine more delayed downstream events subsequent to MC3-R activation. RAW264.7 cells were used and initially validated for their expression of MC3-R message and protein. Confirming previous studies with monocytic cell lines (13, 32), RAW264.7 cells expressed MC1-R mRNA. However, we could not find the MC4-R mRNA, thus allowing us to use the mixed MC3/4-R agonist MTII (31) for most of the subsequent experiments. Because elevated cAMP levels have been associated with HO-1 induction in rat hepatocyte cultures (25), we monitored expression of this and other stress proteins in RAW264.7 cells following incubation with MTII or ACTH. Either peptide provoked a selective and marked up-regulation of HO-1 evident as early as 2–4 h postincubation: this effect was long lasting and fully evident even at the 24-h time point. In full agreement with the study on rat hepatocytes (25), MTII-mediated HO-1 induction was genuinely due to cAMP/PKA signaling, a fact that was confirmed also with primary M{phi} cultures. It is of interest that, during the preparation of this paper, a study showing ACTH induction of HO-1 in a mouse adrenocortical cell line was published (33). It therefore seems that activation of more than one MC-R (certainly MC3-R and MC2-R, the latter being selectively expressed in adrenal cells, and possibly all the other receptors) in target cells can lead to HO-1 up-regulation, thereby making this biochemical link a more general phenomenon. Subsequently, we investigated the potential functional consequences of this induction in experimental inflammatory settings.
In conclusion, this study indicates that HO-1 induction in M{phi} might be a major arm in the complex series of effects that are produced by melanocortin peptides acting at their MC-R. Our analyses on M{phi} function, in the present and previous studies, suggest that MC3-R is the major receptor determinant for transducing the anti-inflammatory actions of these peptides on M{phi}, although it is clear that we could also detect MC1-R in RAW264.7 cells, in analogy to Star et al. (32). However, irrespective of the specific MC-R, a more general picture is emerging in which MC-R activation on the M{phi} cell surface leads to cAMP formation and PKA activation. This signaling pathway produces at least two downstream events: inhibition of cytokine synthesis and release from stimulated M{phi} (in the presence of an inflammogen), which is evident within the first 2 h, and then up-regulation of HO-1 from ≥4 h post-MC-R activation. Importantly, the latter effect is achieved by the melanocortin peptide itself (i.e., in the absence of an inflammogen or M{phi} activator), suggesting that MC-R activation favors the acquisition of the anti-inflammatory proresolving phenotype by the M{phi} (a phenomenon originally described for glucocorticoids (40)). Fig. 8 schematizes this model of two hits, or anti-inflammatory mechanisms, activated by MC3-R agonists.
