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07 August 2010

Cytosolic phospholipase A2 group IVA influence on GM-CSF expression in human lung cells: A pilot study

Milena SokolowskaABCDEFG, Joanna StefanskaBDE, Karolina Wodz-NaskiewiczB, Rafal PawliczakACDEFG

Med Sci Monit 2010; 16(9): BR300-306 :: ID: 881121

Abstract

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important mediator in the differentiation, maturation, and survival of inflammatory cells. It might play a significant role in the pathogenesis of asthma. We have shown previously that cytosolic phospholipase A2 group IV A (cPLA2α) is able to activate gene expression, through peroxisome proliferator-activated receptor (PPAR)-γ response elements (PPRE). In the promoter regions of GM-CSF gene (CSF2), we have found potential PPRE. The goal of the current study was to investigate the influence of cPLA2 overexpression and activation on CSF2 gene expression in human lung cells.
Material/Methods: Subconfluent A549 cells were transfected with GM-CSF reporter gene and cPLA2α overexpression vector. Transfected cells were treated with or without calcium ionophore, A23187, or epidermal growth factor (EGF). Cell lysates were collected and assayed for dual luciferase activity. Some cultures were preincubated with a potent cPLA2α inhibitor, methyl arachidonyl fluorophosphonate (MAFP); a secretory phospholipase A2 inhibitor, thioetheramide-PC; a calcium-independent phospholipase A2 inhibitor, bromoenol lactone (BEL); or vehicle. After preincubation, cells were treated with A23187. Expression of GM-CSF messenger RNA (mRNA) was measured using real-time polymerase chain reaction mRNA quantification.
Results: Overexpression of cPLA2α or activation of endogenous cPLA2α by calcium ionophore or EGF caused a significant increase in GM-CSF relative luciferase activity. Calcium ionophore significantly increased GM-CSF mRNA in A549 human lung cells. These effects were at least in part inhibited by MAFP treatment, but not by BEL or thioetheramide-PC.
Conclusions: These preliminary data might suggest an influence of cPLA2α on GM-CSF gene expression in human lung cells, which could play a role in the pathogenesis of asthma and other inflammatory diseases.

Keywords: Lung - pathology, Group IV Phospholipases A2 - physiology, Granulocyte-Macrophage Colony-Stimulating Factor - metabolism, Genes, Reporter, Gene Expression Regulation, Neoplastic - drug effects, Epidermal Growth Factor - pharmacology, Enzyme Inhibitors - pharmacology, Animals, Lung Neoplasms - pathology, Pilot Projects, Rats, Time Factors

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Medical Science Monitor eISSN: 1643-3750