Regular paperNF-κB activation is inhibited in human pulmonary epithelial cells transfected with α-melanocyte-stimulating hormone vector
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
References (30)
- et al.
Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA
FEBS Lett
(1992) - et al.
Molecular cloning of a novel receptor
J Biol Chem
(1993) - et al.
Molecular cloning, expression, and gene localization of a fourth receptor
J Biol Chem
(1993) - et al.
α-MSH peptides inhibit acute inflammation induced in mice by rIL-1β, rIL-6, rTNF-α and endogenous pyrogen but not that caused by LTB4, PAF and rIL-8
Cytokine
(1992) - et al.
α-Melanocyte-stimulating hormone inhibits NF-κB activation and IκBα degradation in human glioma cells and in experimental brain inflammation
Exp Neurol
(1999) - et al.
Anti-inflammatory actions of the neuroimmunomodulator α-MSH
Immunol Today
(1997) - et al.
Regulatory elements involved in Tax-mediated transactivation of the HTLV-1 LTR
Virology
(1993) - et al.
Function and activation of NF-κB in the immune system
Annu Rev Immunol
(1994) The NF-κB and IκB proteinsnew discoveries and insights
Annu Rev Immunol
(1996)- et al.
Cloning and expression of a new member of the melanocyte-stimulating hormone receptor family
J Mol Endocrinol
(1994)
Control of IκB-α proteolysis by site-specific, signal-induced phosphorylation
Science
α-Melanocyte stimulating hormone in the modulation of host reactions
Endocr Rev
Regulation of tumor necrosis factor (TNF) alpha transcription in macrophagesinvolvement of four κB-like motifs and of constitutive and inducible forms of NF-κB
Mol Cell Biol
Characterization of a functional NF-κB site in the human interleukin 1β promotorevidence for a positive autoregulatory loop
Mol Cell Biol
Double-stranded RNA-dependent protein kinase activates transcription factor NF-κB by phosphorylating IκB
Proc Natl Acad Sci USA
Cited by (19)
Targeting melanocortin receptors as potential novel therapeutics
2006, Pharmacology and TherapeuticsCreation of the Gain-of-Function Mutation of the MITF Gene Related to Melanogenesis Using the CRISPR-Cas9 System
2022, Russian Journal of GeneticsMelanocortin Regulation of Inflammation
2019, Frontiers in Endocrinology