Cell wounding of primary mouse keratinocytes ‐ Role of PLD

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Skin is the largest organ in the body and is the first barrier of defense against environmental insults. Wounding of the keratinocytes, the major cell type of the epidermis of the skin, is a routine phenomenon but repair involves complex downstream signaling events. We hypothesized that wounding of primary mouse keratinocytes would activate phospholipase D (PLD) and that this activation would be decreased by curcumin. Activated PLD catalyzes the hydrolysis of phospholipids to produce phosphatidic acid (PA), which can be dephosphorylated by lipid phosphate phosphatases to produce diacylglycerol (DAG). Primary mouse keratinocytes were pre-labeled with [3H] oleic acid for 20–24 hours. Wounding was induced by scraping the cells from the dish. As a control for loss of adhesion, cells were also treated with 100 μl of trypsin. Ethanol at a concentration of 0.5 % was added to take advantage of the transphosphatidylation reaction catalyzed by PLD as a means of measuring PLD activity. Lipids were extracted into chloroform/methanol after 15 minutes, separated by thin-layer chromatography, and radioactivity in phosphatidylethanol (PEt) was quantified using a scintillation counter. As we hypothesized, wounding of primary mouse keratinocytes led to activation of PLD and our results suggest that this activation was decreased by curcumin pre-treatment. We propose that wound repair may involve activation of PLD, and curcumin may reduce this activation thus decreasing injured cell survival. Experiments have also been performed to examine the time course of PLD activation and whether this signaling event is mediated by PLD1 and /or PLD2. Research is in progress to determine the role of wounding-induced PLD activation in wound repair.