062 - Regulation of MUC5AC expression by arachidonic acid metabolites - 05/12/08

Background: Cytosolic phospholipase A2 (cPLAb2) is a rate- limiting key enzyme controlling the release of arachidonic acid (AA) from membrane phospholipids. Conversion of AA by cyclooxygenases (COX) and lipoxygenases (LOX) generates prostaglandins and leukotrienes, respectively. These AA metabolites play an important role in the control of inflammation. We have previously observed that cPLA2 is involved in mucus hyper-secretion and expression of the mucin Muc5ac, in mice deficient in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (Dif et coll. submitted). Muc5ac is a major mucin present in bronchial secretions of CF patients (Thorax. 2007. 62:153-61)

Aim: Identify the signaling pathways involved in the mucin Muc5ac expression mediated by cPLA2 in the bronchial epithelial cell line NCI-H292. We also evaluated the effect of an inhibitor of CFTR chloride channel function (Inh172 inhibitor) on Muc5ac expression.

Methods: Production of Muc5ac was measured in cell lysates and supernatants by specific ELISA, after stimulation with PMA or TGF-alpha. cPLA2 activity was analyzed in cell lysates, after hydrolysis of phosphatidyl choline labeled with radioactive AA. The role of cPLA2, COX and LOX in Muc5ac production was examined after treatment with specific inhibitors. Activation of transcription factors was followed using EMSA.

Results: Both PMA- and TGF-alpha induced Muc5ac expression were mimicked by addition of exogenous AA and inhibited after pre-treatment of the cells with the cPLA2 inhibitor MAFP. Further, Muc5ac expression was inhibited by a LOX inhibitor (NDGA) but not by COX inhibitors (aspirin, NS398). cPLA2 induced Muc5ac expression though PPAR-β-dependent process. Indeed, MAFP abrogated PPAR-β activation and PPAR-β agonists induced Muc5ac production. However, NF-kB inhibition failed to interfere in Muc5ac production. On the other hand, Inh172 inhibitor failed to increase Muc5ac production in stimulated cells.

Conclusion: cPLA2 plays an important role in the production of Muc5ac mucin. This involves LOX metabolites rather than COX, and a signaling pathway resulting in the activation of the transcription factor PPAR-β. In addition, we showed that induced Muc5ac secretion does not depend on CFTR chloride channel function.

Acknowledgments: Work supported by “Vaincre la Mucoviscidose”