Inhibition of NF-κB activity by plasmid expressed αMSH peptide

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Abstract

α-Melanocyte Stimulating Hormone (αMSH) is a neuroimmunomodulatory peptide with remarkable anti-inflammatory properties. Daily or twice daily administration of the peptide reduces the symptoms of several inflammatory animal disease models and the peptide has demonstrated safety in human trials. Unfortunately, the pharmacokinetics of peptide delivery are not favorable from the pharmaceutical perspective. For this reason, plasmid-based vectors were created that constitutively express the immunomodulatory peptide. The fusion constructs encode the 13 amino acids of αMSH in frame with the first domain of serum albumin, separated by a linker and furin cleavage sites. The fusion proteins were expressed and processed in human fetal kidney (293) cells. Supernatant from B16/F10 cells transfected with the constructs stimulated secretion of melanin from melanocytes. Furthermore, transfected cytoskeletal muscle (Sol8) cells secreted bioactive αMSH that reduced NF-κB-mediated transcriptional activation of a luciferase reporter gene. The activity of these vectors provides tools and the impetus for testing the constructs in several animal models of chronic inflammation.

Introduction

α-Melanocyte Stimulating Hormone (αMSH) is a 13-amino acid neuroimmunomodulatory peptide that arises through proteolytic processing of the proopiomelanocortin precursor molecule (POMC). αMSH is produced in several tissues, including the pituitary gland, gut, brain, circulation, and skin Bateman et al., 1989, Catania and Lipton, 1993, Lipton and Catania, 1997, Luger et al., 1997. αMSH plays a role in the control of melanogenesis, endocrine metabolism, and has strong anti-inflammatory effects Lipton and Catania, 1997, Smith and Funder, 1988, all of which are mediated via interaction with melanocortin receptors (MC-R). αMSH-stimulated melanogenesis results from binding of the peptide to one of the MC-Rs on the surface of melanocytes, which is followed by a rapid increase of cyclic AMP, and activation of tyrosinase, one of the key enzymes for the production of melanin De Graan et al., 1987, Sheppard et al., 1983, Wong and Pawelek, 1973. POMC-derived peptides also play an important role in energy homeostatis: mutations in the αMSH precursor POMC gene in both mice and humans have been implicated in the occurrence of obesity Krude et al., 1998, Yaswen et al., 1999 and MC-Rs have been implicated in control of food intake and body weight Huszar et al., 1997, Ollmann et al., 1997. Finally, binding of αMSH to the melanocortin receptors inhibits the production and activity of pro-inflammatory cytokines Gantz et al., 1993, Mountjoy et al., 1992.

The anti-inflammatory activity of αMSH has been the subject of many recent studies. The peptide's anti-inflammatory actions are mediated via a family of specific G-protein coupled melanocortin receptors Gantz et al., 1993, Mountjoy et al., 1992. Binding of αMSH to the MC-Rs activates adenylate cyclase (Mertz and Catt, 1991), which results in an increase of cAMP in the cytoplasm. In turn, this event modulates a signal transduction pathway that prevents dissociation of the IκB–NFκB complex; thus averting translocation of NFκB into the nucleus and the subsequent transcription of proinflammatory cytokines such as TNFα, IL-1, IL-6, and IL-8 Collart et al., 1990, Hiscott et al., 1993, Kunsch et al., 1994, Libermann and Baltimore, 1990, Matsusaka et al., 1993, Roulston et al., 1995. The anti-inflammatory activity of αMSH has been documented in a wide variety of cells that express the MC-Rs; αMSH inhibits the ability of macrophages and monocytes to produce nitric oxide (NO) and TNFα, Becher et al., 1999, Chiao et al., 1996, Taherzadeh et al., 1999, and it prevents cytokine production by fibroblasts, neutrophils, keratinocytes, and brain tissue Bohm et al., 1999, Brzoska et al., 1999, Ichiyama et al., 1999a, Robertson et al., 1986.

The αMSH peptide is a powerful drug candidate due to its pleiotropic effects on inflammation and energy homeostatis, and has potential therapeutic applications for the treatment of inflammatory diseases and obesity. It may be possible to utilize the broad therapeutic properties of αMSH to create gene-based drug treatments as a means of inhibiting chronic inflammatory aspects of autoimmune disease such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), or multiple sclerosis (MS), and regulating feeding behavior and body weight. To alleviate the potential difficulties of small peptide expression from plasmid-based vectors, and to enhance the expression of bioactive αMSH, we have designed constructs that encode the 13 amino acids of αMSH in frame with the first domain of serum albumin. Serum albumin is the most abundant protein in the circulatory system and has been successfully utilized as a carrier for protein delivery (Fiume et al., 1988). Our results show that the recombinant αMSH proteins are expressed and secreted by transfected cells. The secreted peptide is bioactive and most importantly can inhibit NF-κB transcriptional activation of a reporter gene.

Section snippets

Construction of expression vectors

Sequences coding for the signal peptide, propeptide, and the first 195 amino acids of human and mouse serum albumin were amplified by RT PCR from total RNA from either HepG2 cells or mouse liver, respectively. Total RNA was extracted from HepG2 cells using the RNeasy kit (Qiagen, Valencia, CA). Mouse liver was homogenized using Trizol (Life Technologies, Grand Island, NY) and total RNA was isolated using the RNeasy Mini Kit (Qiagen). The integrity of the RNA was examined by agarose gel

Design of αMSH expression vectors

In order to promote secretion of a bioactive αMSH peptide from cells transfected with αMSH encoding plasmids, fusion genes were created that encode αMSH downstream and in frame with a non-immunogenic and highly expressed protein, serum albumin. A linker was introduced at the albumin-αMSH junction, and a furin cleavage site was engineered between the linker and αMSH sequences (Fig. 1). The albumin domain of the fusion protein acts as a carrier for the αMSH peptide, as it passes through the

Discussion

We have created plasmid-based vectors that express a bioactive form of the αMSH peptide in vitro. The amino acid sequence of the αMSH peptide was fused in frame to the first domain of a carrier protein that is neither toxic nor immunogenic, serum albumin, followed by a linker and a furin cleavage site. Our results show that albumin-αMSH fusion proteins are expressed and processed as expected. Western analysis suggests that the albumin domain of the fusion construct is removed from the αMSH

Acknowledgements

We thank Janet Lathey and Thomas Luby for critical reading of the manuscript.

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