Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes.
The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A2 (PLA2), which catalyzes the release of sn-2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA2 in mouse keratinocytes, a cytosolic or cPLA2 and two secretory or sPLA2s in the membrane. The PLA2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA2 and at least one type of sPLA2. Inhibitor data further confirmed the identities of these PLA2s. The cPLA2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4alpha-TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187- and 4alpha-TPA-treated keratinocytes suggests activation of sPLA2. These activities may be due to the existence of both type I and type II sPLA2, as both were identified by polymerase chain reactions. In conclusion, keratinocytes express several forms of phospholipase A2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.
Li-Stiles
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The University of Texas M.D. Anderson Cancer Center, Science Park, Research Division, Smithville 78957, USA.
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Fischer
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D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D000078404:Epidermal Cells; D006358:Hot Temperature; D007527:Isoenzymes / Q000032:analysis*; D015603:Keratinocytes / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D019787:Linoleic Acid / Q000378:metabolism; D051379:Mice; D019301:Oleic Acid / Q000378:metabolism; D010741:Phospholipases A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D012333:RNA, Messenger; D016718:Arachidonic Acid; D019301:Oleic Acid; D000001:Calcimycin; D019787:Linoleic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium