Abstract
Lysophosphatidic acid (LPA) is a receptor-active lipid mediator with a broad range of biological effects. Ovarian cancer cells synthesize LPA, which promotes their motility, growth, and survival. We show that a murine homolog of a human protein previously reported to hydrolyze LPA is a highly selective detergent-stimulated LPA phosphatase that can be used to detect and quantitate LPA. Use of this protein in novel enzymatic assay demonstrates that SK-OV-3 ovarian cancer cells release physiologically relevant levels of biologically active LPA into the extracellular space. LPA release is markedly increased by nucleotide agonists acting through a P2Y4 purinergic receptor. Promotion of LPA formation by nucleotides is accompanied by stimulation of phospholipase D (PLD) activity. Overexpression of both PLD1 and PLD2 in SK-OV-3 cells produces active enzymes, but only overexpression of PLD2 results in significant amplification of both nucleotide-stimulated PLD activity and LPA production. SK-OV-3 cells express and secrete a phospholipase A2 activity that can generate LPA from the lipid product of PLD, phosphatidic acid. Our results identify a novel role for nucleotides in the regulation of ovarian cancer cells and suggest an indirect but critical function for PLD2 in agonist-stimulated LPA production.
Highlights
Lysophosphatidic acid (LPA) is a receptor-active lipid mediator with a broad range of biological effects
LPA can be generated by phospholipase A (PLA)-catalyzed hydrolysis of phosphatidic acid (PA), which can be formed in stimulated cells through actions of inositol lipid-specific phospholipase C (PLC), diacylglycerol kinase, or phosphatidylcholine-specific phospholipase D (PLD) [11]
Together with our demonstration that adenovirus-expressed PLD1 is active in SK-OV-3 cells when measured in vitro, these results suggest that the apparent preference shown by the P2Y4 receptor for activation of PLD2 activity does not arise from some undefined impairment of recombinantly expressed PLD1
Summary
Lysophosphatidic acid (LPA) is a receptor-active lipid mediator with a broad range of biological effects. Use of this protein in novel enzymatic assay demonstrates that SK-OV-3 ovarian cancer cells release physiologically relevant levels of biologically active LPA into the extracellular space. SK-OV-3 cells express and secrete a phospholipase A2 activity that can generate LPA from the lipid product of PLD, phosphatidic acid. LPA can be generated by phospholipase A (PLA)-catalyzed hydrolysis of phosphatidic acid (PA), which can be formed in stimulated cells through actions of inositol lipid-specific phospholipase C (PLC), diacylglycerol kinase, or phosphatidylcholine-specific phospholipase D (PLD) [11]. Studies with platelets implicate membrane microvesicles released in response to agonist activation as key intermediates in LPA production, but the relevance of this pathway to the process of LPA production by other cell types is unknown [4]
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