Elsevier

Peptides

Volume 21, Issue 10, October 2000, Pages 1473-1477
Peptides

Regular paper
NF-κB activation is inhibited in human pulmonary epithelial cells transfected with α-melanocyte-stimulating hormone vector

https://doi.org/10.1016/S0196-9781(00)00300-4Get rights and content

Abstract

α-Melanocyte-stimulating hormone (α-MSH) modulates inflammation. We investigated the influence of α-MSH on NF-κB activation in human pulmonary epithelial cells (A549) using a plasmid vector encoding α-MSH (pCMV-ssMSH). Electrophoretic mobility shift assays demonstrated that NF-κB activation induced by lipopolysaccharide was inhibited in A549 cells transfected with pCMV-ssMSH. Western blot analysis revealed that this inhibition was linked to preservation of expression of IκBα protein. Chloramphenicol acetyltransferase assay indicated that NF-κB-dependent reporter gene expression was suppressed in A549 cells transfected with pCMV-ssMSH. The findings indicate that anti-inflammatory actions are exerted via modulation of NF-κB activation by preservation of IκBα protein in human pulmonary epithelial cells transfected with α-MSH vector. We showed a possibility of gene therapy for chronic inflammatory lung diseases.

Introduction

Inflammation is an important part of the pathogenesis of pulmonary diseases, not only in infectious diseases due to bacteria, viruses, or fungi, but also in chronic obstructive pulmonary disease and neonatal chronic lung disease [24], [27]. Inflammation mediated by proinflammatory cytokines is associated with and promotes the pathogenesis of these disorders. It is therefore important to modulate pulmonary inflammation in treatment of patients with these lung disorders.

α-Melanocyte-stimulating hormone (α-MSH) is a pro-opiomelanocortin derivative which shares the 1–13 amino acid sequence with adrenocorticotropic hormone; this peptide occurs in the pituitary, brain, skin, circulation, and other sites [5]. α-MSH is a melanocortin and five melanocortin receptor subtypes have been identified (MC1R through MC5R) [3], [6], [8], [9], [20]. α-MSH modulates inflammation [16]. α-MSH inhibits production of proinflammatory cytokines, nitric oxide, prostaglandin E2, and neopterin [10], [22], [23], [28], [29].

Nuclear factor kappa B (NF-κB) is a pivotal transcription factor. The target genes encode proinflammatory cytokines such as IL-1, IL-6, IL-8 and TNF-α [7], [11], [14], [15], [19], [25]. The NF-κB is normally inactive, bound by members of the IκB family, including IκBα, in the cytoplasm [1], [2]. Phosphorylation of IκBα by drugs, cytokines, bacterial products and viruses leads to IκBα degradation, translocation of NF-κB to the nucleus, and transcription of proinflammatory cytokine genes [4], [13]. Exogenous α-MSH inhibits NF-κB activation and IκBα degradation in human monocytic leukemia cells and glioma cells and in murine experimental brain inflammation [12], [18].

We tested the hypothesis that α-MSH inhibits NF-κB activation in human pulmonary epithelial cells (A549) using a plasmid vector encoding α-MSH (pCMV-ssMSH).

Section snippets

Construction of pCMV-ssMSH expression vector

The strategy used for construction of the pCMV-ssMSH chimeric gene involved the initial synthesis by PCR of a fragment spanning nucleotides 33 to 120 (Genbank Accession No. J03783) of the murine IL-6 cDNA (kindly provided by Dr. Van Snick, Belgium) corresponding to the complete signal sequence for this gene. In order to get secretion of MSH from the cell we utilized the signal sequence from the murine IL-6 gene. A fusion gene was constructed (as I indicated to you when I originally provided the

Results

The α-MSH concentrations of culture fluid from cells transfected 48 h earlier with pCMV-ssMSH were significantly greater than those of cells with control transfections (33.4 ± 5.1 pmol/liter vs 6.9 ± 0.8 pmol/liter) (Fig. 2). EMSA demonstrated that NF-κB activation induced by LPS was suppressed in cells transfected with pCMV-ssMSH compared with activation in cells transfected with pCMV (Fig. 3). Densitometry showed an inhibition of approximately 45% at 1 h, and 55% at 2 h, in cells

Discussion

EMSA revealed that translocation of NF-κB to the nucleus was inhibited in pCMV-ssMSH transfected A549 cells. The CAT assay indicated that transcription linked to NF-κB was inhibited in pCMV-ssMSH transfected A549 cells. Western blot analysis demonstrated that these inhibitions were linked to the preservation of IκBα protein. α-MSH concentration was significantly elevated in the medium from pCMV-ssMSH transfected A549 cells. Therefore α-MSH produced in pCMV-ssMSH transfected A549 cells exerts

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