#58 Unravelling the Signaling Pathways of Oxidized Phosphatidylcholine in Primary Human Airway Smooth Muscle Cells
Mirna MK Ragheb, University of Manitoba; Jason Kindrachuk, Univeristy of Manitoba; Andrew J Halayko, University of Manitoba and Children’s Hospital Research Institute of Manitoba; Christopher D Pascoe, University of Manitoba and Children’s Hospital Research Institute of Manitoba
Oxidative stress is a key determinant of asthma severity and correlates with acute asthma exacerbations. Generated through oxidative stress, oxidized phosphatidylcholine (OxPC) induces inflammation in human airway smooth muscle cells (HASM) by increasing COX-2 expression and mediating the release of cytokines and oxylipins. However, the mechanism of OxPC signaling is unknown. Therefore, we hypothesize that OxPC increases COX-2 gene expression through a receptor-mediated process.
Primary HASM (non-COPD donors, n=3) were grown and serum-starved for 96 hours. Cells were pre-incubated with specific inhibitors for 2 hours, then exposed to OxPC (80 µg/mL) for 24 hours. We tested inhibitors for Prostaglandin E receptors (EP), platelet activating factor receptor (PAFR), CD36, Toll-like receptors 2/4 (TLR 2/4), mitogen-activated protein kinase kinase 1/2 (MEK1/2), protein kinase C α/β1 (PKC), and rho-associated, coiled-coil containing protein kinase (ROCK1/2). RNA was collected for qPCR 24 hours post-treatment. Additionally, cell lysates from HASM exposed to OxPC and PSPC (control phospholipid) for 6 hours were collected to perform kinomics analysis.
Inhibitors for EP, CD36, PAFR, and TLR 2/4 receptors were unable to prevent the increase in COX-2 gene expression induced by OxPC. Additionally, inhibitors for MEK1/2 and ROCK1/2 (10µM) were unable to attenuate the OxPC-induced COX-2 gene expression. The inhibitor for PKC (10µM) significantly reduced OxPC-induced COX-2 gene expression by 3-4-fold (p<0.05). Moreover, the OxPC-induced kinase activity from the kinomics study suggested a strong correlation between OxPC and MyD88-dependent signaling and overall TLR signaling, which might not be specific for only TLR2 or TLR4.
OxPC-induced COX-2 gene expression does not require the activation of EP1/2, PAFR, CD36 or TLR 2/4 receptors in HASM; however, the signaling cascade appears to be dependent on PKC activity. Our kinomics data suggests that TLR signaling is involved in responding to OxPC but it is unknown if these receptors play a role in COX-2 induction.