Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling

Abstract

Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid, and there is currently much interest in elucidating messenger functions for this molecule. We report here that wounding sheets of corneal epithelial and Madin Darby canine kidney cells induces strong activation of phospholipase D, and we provide evidence that activation is amplified through a positive feed-back loop. Short-chain analogues of phosphatidic acid induce motility robustly in corneal and other epithelial cell types. The effects of these analogues were not the result of their conversion to the corresponding diacylglycerol or lysophosphatidic acid, implying that phosphatidic acid acts directly on one or more cellular targets. Strikingly, phosphatidic acid signaling was found to stimulate the epidermal growth factor receptor (EGFR) through a transactivation process. Healing of wounds in sheets of corneal epithelial cells is absolutely dependent on epidermal growth factor receptor signaling, and the present data suggest that its activation is a result of wound-induced phospholipase D activation.

The Molecular Basis of Phospholipase D2-Induced Chemotaxis: Elucidation of Differential Pathways in Macrophages and Fibroblasts

“…We have studied the mechanism of PLD-induced chemotaxis and found that the enzymatic activity of the lipase needs to be intact. This is in agreement with the findings of Mazie et al (43), who demonstrated that EGF triggers the activation of PLD in MDCK cells. PA, the product of PLD activation, is a critical mediator of EGF-dependent cell motility.…”

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…Wounded corneal epithelial cells undergo an EMT as cells migrate at the leading edges of the wound. Treatment with exogenous PtdOH or overexpression of PLD2 caused a dramatic reduction of cell surface E-cadherin in these cells (Mazie et al, 2006). However, butanol did not decrease EGF stimulation of ERK in this system, suggesting that PLD may regulate E-cadherin through different means.…”

“…As such, butanol treatment or knockdown of PLD is associated with defects in the ability to form adhesions (Aguirre Ghiso et al, 1997;Iyer et al, 2006). The speed of migration is largely controlled by the speed at which cells form and detach from these adhesions (Lauffenburger and Horwitz, 1996) and PLD regulates migration speed in wound-healing assays (Mazie et al, 2006). PLD activity is associated with an increase in vinculin levels (Mazie et al, 2006) as well as activation of focal adhesion kinase, a key regulator of focal adhesion turnover (Lli c et al, 1995;Knoepp et al, 2008).…”

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling

“…1,2-Dioctanolyl-glycero-3-phosphate (C 8 -PA) is a water-soluble synthetic phosphatidic acid that traverses the plasma membrane and mimics the effects of phospholipase D expression (Mazie et al, 2006). We added C 8 -PA to the media of HeLa cells at 24 h after HeLa cells were either untreated (Cells), exposed to Lipofectamine RNAiMAX alone (Lipo) or transfected with scrambled control (Ctrl siRNA) or choline kinase-a-specific siRNA (CK siRNA) for 48 h. The cells were then examined for (a) choline kinase-a and choline kinase-b mRNA expression using real-time reverse transcriptase (RT)-PCR (normalized to b-actin), (b) choline kinase protein expression using western blot and (c) phosphocholine (PCh) using nuclear magnetic resonance (NMR).…”